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Lecture Topics by Day
Day 1 (3h)
Introduction to protein chemistry
Strategies used by enzymes to accelerate reaction rates
Day 2 (3h)
Protein stability elucidated and enhanced via protein engineering
Protein folding & unfolding probed via protein engineering
Day 3 (2.5h)
Protein folding and unfolding probed via protein engineering (continued)
Lecture Series
Special Topics in
Protein Chemistry
(equivalent to a 2credict course)
Day 4 (3h)
The combined power of in vitro chemical modification and paper-supported chromatography as a probe
of structure and function
Day 5 (3h)
The combined power of in vitro chemical modification and paper-supported chromatography as a probe
of structure and function (continued)
In vitro manipulation of protein monomers or their environment to enhance performance
Day 6 (2.5h)
In vitro manipulation of protein monomers or their environment to enhance performance (continued)
A closer look at optimizing protein function in non-aqueous environments
Protein purification and related analytical methods
Short examination scheduling
Lecturer: Alpay Taralp, Materials Science & Engineering Program, Sabancı University,
Istanbul 34956; [email protected]; http://people.sabanciuniv.edu/~taralp/
© 2006, Alpay Taralp, Sabanci University
Introduction to Protein Chemistry
© 2006, Alpay Taralp, Sabanci University
References Relevant to this Material
1. Lundblad, R.L., Techniques in protein modification, CRC Press, 1995, 0-8493-2606-0
2. Wong, S.S., Chemistry of protein conjugation and crosslinking, CRC Press, 1991, 0-8493-5886-8
3. Nagradova, N.K., Lavrik, O.I., Kurganov, B.I., Chemical Modification of Enzymes, Nova Science, Inc., 1995, 1-5607-2238-X
4. Brown, W.E., Howard, G.C., Practical Methods in Advanced Protein Chemistry, CRC Press, 2000, 0-8493-9453-8
5. J.M.Walker, Ed., Protein protocols on CD-ROM, Humana Press, 1998, 0-89603-514-X
6. Darbre, A., Practical Protein Chemistry: A Handbook, John Wiley and Sons, 1986.
7. Eyzaguirre, J., Chemical Modification of Enzymes: Active Site Studies, Prentice Hall, 1987, 0-47020-763-9
8. Methods in Enzymology Series, Academic Press,Vols.11, 25-27, 47-49, 61, 91, 117, 130, 131, 135-137.
9. Glazer, A.N, Delange, R.J., Sigman, D.S., Chemical Modification of Proteins, Elsevier Science, 1975, 0-44410-811-4
10. Feeney, R.E., Whitaker, J.R., American Chemical Society Advances in Chemistry Ser. (No. 160) - Food Proteins: Improvement
Through Chemical & Enzymatic Modification, Books on Demand, 0-31710-649-X
11. Feeney, R.E., Whitaker, J.R., Modification of Proteins: Food, Nutritional & Pharmacological Aspects, 1982, 0841206104 Advances in
Chemistry Ser. (No. 198) American Chemical Society
12. Feeney, R.E., Whitaker, J.R., Protein Tailoring & Reagents for Food & Medical Uses, Marcel Dekker Incorporated, 1986, 0-82477616-X.
13. Bailey, J. L., Techniques in Protein Chemistry, Elsevier Publishing Company, 1962, Lib. Congress 62-19691.
14. Lundblad, R. L., Chemical Reagents for Protein Modification, 2nd Ed., CRC Press, 1991, 0-8493-5097-2.
15. Walsh, G., Headon, D.R., Protein Biotechnology, John Wiley and Sons, 1994, 0-471-94393-2.
16. Mean, G., Feeney, R.E., Chemical Modification of Proteins, Holden Day, Inc., 1971, Lib. Congress 74-140785.
17. McGrath, K., Kaplan, D., Protein-Based Materials, Birkhauser, 1996, 0-8176-3848.
18. Koskinen, A.M.P. and Klibanov, A.M., Enzymatic Reactions in Organic Media, Blackie Academic and Professional, 1996, 0-75140259-1.
19. Suckling, C.J., Gibson, C.L., Pitt, A.R., Enzyme Chemistry: Impact and Applications, Blackie Academic and Professional, 1998, 07514-0362-8.
20. Magdassi, S., Surface Activity of Proteins: Chemical and Physicochemical Modifications, Marcel Dekker, Inc., 1996, 0-8247-9532-6.
21. Rawn, J. D., Proteins, Energy and Metabolism, Neil Patterson Publishers, 1989, 0-89278-404-0.
22. Fersht, A.R., Structure and Mechanism in Protein Science, W.H. Freeman and Company, 1999, 0-7167-3268-8.
23. Oxender, D.L., Fox, F.C., Protein Engineering, Alan R. Liss, Inc., 1987, 0-8451-4300-X.
24. Crieghton, T.E., Protein Structure: A Practical Approach, 2nd Edition, Oxford University Press, 1997, 0-19-963618-4.
25. Crieghton, T.E., Proteins: Structures and Molecular Properties, 2nd Edition, W.H. Freeman and Company, 1993, 0-7167-2317-4.
26. Wüthrich, K., NMR of Proteins and Nucleic Acids, John Wiley and Sons, 1986, 0-471-82893-9.
27. Journals focused on the subject of protein chemistry: Journal of Protein Chemistry; Protein Science; Biochemistry; Journal of
Biological Chemistry; Biomacromolecules
28. Catalogues! Promega Protein Guide: Tips and Techniques; Pierce Products; Biorad Life Science Research Products
© 2006, Alpay Taralp, Sabanci University
The way to operate True or false? A student of protein chemistry…
1. buys the best possible instrument and then tries to force the problems of
protein chemistry to suit the use of the machine.
2. works in a problem-oriented manner in which experience and knowledge are
adopted to accommodate available machines.
3. relies first on imagination, then knowledge, then machines (Consider the
contrast between H. Noyrath vrs. B. Hartley). What was one of Einstein's
quotations?
4. believes that protein investigation is as simple and amusing as watching
Indiana Jones running away from a band of sword-wielding bandits (The
Okum's Razor argument).
5. should use all his/her time reading primary references and never use his/her
own ideas, intuitions or beliefs
6. gives more credit to the ideas of a supervisor than to their own ideas
© 2006, Alpay Taralp, Sabanci University
To study proteins is to study diversity! i.e., diversity
of structure, function, chemistry, analysis, etc.
To emphasize the scope of diversity, let us focus on
structural diversity...
• Structure is a shape, sequence, order, orientation,
configuration, etc. of an atom or molecule.
• Eg. The electronic structure of carbon is 1s22s22p2.
Cl
Cl C Cl
Cl
• Eg. CCl4 has a tetrahedral shape.
• Eg. The primary structure of insulin begins with:
• Eg. The tertiary structure of cytochrome c is globular.
Diversity of structure: Static vs. dynamic
• Structure (an other traits) may be static (fixed)
or dynamic (changing) over time.
• The time frame of structural change may be
very long (the half life of 238U is 4.5x109 years)
or very brief (a 10 fs chemical interaction)
-
I-CH3 OH
(
U238
shorter) Life-time of event (longer
Ra226
)
We must characterize structural diversity
to understand proteins
• Question 1: Can you see the 3-D
shape of myoglobin with your eyes?
• Question 2: Can you live 4.5 billion
years to see ½ of the 238U decay?
• Question 3: Can you react quickly
enough to measure a chemical
interaction?
The answer to each is NO!
>> So we must use machines...Why?
Reason one: We are limited by resolving
power…If our information carrier is visible
light, we are limited to an approximate
resolution of 0.2mm. Details smaller than
0.2mm are lost.
500nm
Look Wilma, what
a nice smooth
surface!! Light
you see
actual surface
Reason two: The event is often faster than the
speed of the measurement…You obtain a blurred
average.
• Eg. Try photographing chicks in a bowl
a.
b.
c.
Nature features an invisible world
of details & diversity. Instruments
allow us to see these details…
Balloons pierced with a bullet
Dynamic changes along an aqueous
surface: Droplets captured in motion
A Look at Diverse Protein Structures
1. Protein structure is not rigid!
2. Protein structure bears many aspects
Proteins are generally made from
20 types of amino acids, which are:
H2N
OH
R H
>> Linked by amide bonds (rarely: ester
bonds, Ser/Thr; thioester bonds, Cys)
>> Bridged via –S-S- groups or the
desmosine group, a 4-lys crosslinker
>> Enzymatically processed:
hydroxylated, formylated,
phosphorylated, glycosylated,
amidated, sulfonated, acetylated,
methylated, hydrolyzed, etc.
S
O
S
>> Associated to non-proteins, e.g.,
WATER, heme groups, etc.
© 2006, Alpay Taralp, Sabanci University
I am dynamic!
Linking the building blocks: Stereoelectronic properties of
the peptide bond
O
O
O
O
O
H2N
OH
C
H2N
OH
R H
N
H
H
C
H
H
H
H
R H
N
C
H
N
H
H
The peptide bond (like formamide, below & above right) is stabilized by resonance: 60%
amide and 40% hydroxyimine character
O
O
C
H
N
H
C
H
N+
H
H
H
What else do we observe?
All 6 atoms lie in the same plane, i.e., the peptide bond is planar.
p-electrons are distributed over the C-O and C-N bonds.
The C-N bond is 10% shorter than a normal C-N bond.
The peptide bond is trans.
C
O
C
O
+
N C
N C
H
H
C
C
The peptide bond has a permanent dipole (m = 3.7D)
© 2006, Alpay Taralp, Sabanci University
Protein diversity is enabled by linking diverse building blocks!
Stereoelectronic differences of common amino acid residues:
Amino Acid
Let. Codes
MW
Surface Ǻ2
Volume Ǻ3
pKa, Side,25°C
pI, 25°C
Sol., g/100g
Crys. d, g/ml
Alanine
ALA
A
71.09
115
88.6
-
6.107
16.65
1.401
Arginine
ARG
R
156.19
225
173.4
~12
10.76
15
1.1
AsparticAcid
ASP
D
115.09
150
111.1
4.5
2.98
0.778
1.66
Asparagine
ASN
N
114.11
160
114.1
-
-
3.53
1.54
Cysteine
CYS
C
103.15
135
108.5
9.1-9.5
5.02
v. high
-
GlutamicAcid
GLU
E
129.12
190
138.4
4.6
3.08
0.864
1.460
Glutamine
GLN
Q
128.14
180
143.8
-
-
2.5
-
Glycine
GLY
G
57.05
75
60.1
-
6.064
24.99
1.607
Histidine
HIS
H
137.14
195
153.2
6.2
7.64
4.19
-
Isoleucine
ILE
I
113.16
175
166.7
-
6.038
4.117
-
Leucine
LEU
L
113.16
170
166.7
-
6.036
2.426
1.191
Lysine
LYS
K
128.17
200
168.6
10.4
9.47
v. high
-
Methionine
MET
M
131.19
185
162.9
-
5.74
3.381
1.340
Phenylalanine
PHE
F
147.18
210
189.9
-
5.91
2.965
-
Proline
PRO
P
97.12
145
112.7
-
6.3
162.3
-
Serine
SER
S
87.08
115
89.0
-
5.68
5.023
1.537
Threonine
THR
T
101.11
140
116.1
-
-
v. high
-
Tryptophan
TRP
W
186.12
255
227.8
-
5.88
1.136
-
Tyrosine
TYR
Y
163.18
230
193.6
9.7
5.63
0.0453
1.456
Valine
VAL
V
99.14
155
140.0
-
6.002
8.85
1.230
© 2006, Alpay Taralp, Sabanci University
Different building blocks have stereoelectronic differences:
Some are more similar than others.
Residues joined by solid lines may be replaced with
95% confidence
© 2006, Alpay Taralp, Sabanci University
The “20” amino Acids
Non-polar amino acids
E
E
Charged basic amino acids
E
E
E
E
E*
E*
E
Charged acidic amino acids
Polar uncharged amino acids
E
#21
#22
E*
Stop codon +
special tRNA
Postsynthetic
Selenocysteine
5-Hydroxylysine
Pyrrolysine© 2006, Alpay Taralp,Selenomethionine
4-Hydroxyproline g-Carboxyglutamic acid
Sabanci University
estimatedEffect
hydrophobic following residue
(L) or side-chain burial (R) [kcal/mol]
Amino Acids
in 55 Proteins
SEA >30
Å2
30 > SEA >10
Å2
SEA <10
Å2
Abs. & % nonpolar surface
of residues vs. total Å2
Glutamic acid
0.93
0.03
0.04
69 (36%) vs. 190
1.73
0.5
Lysine
0.93
0.05
0.02
122 (61%) vs. 200
3.05
1.9
Arginine
0.84
0.11
0.05
89 (40%) vs. of 225
2.23
1.1
Asparagine
0.82
0.08
0.10
42 (26%) vs. 160
1.05
-0.1
Aspartic acid
0.81
0.10
0.09
45 (30%) vs. 150
1.13
-0.1
Glutamine
0.81
0.09
0.10
66 (37%) vs. 180
1.65
0.5
Proline
0.78
0.09
0.13
124 (86%) vs. 145
3.10
1.9
Threonine
0.71
0.13
0.16
90 (64%) vs. 140
2.25
1.1
Serine
0.70
0.10
0.20
56 (49%) vs. 115
1.40
0.2
Tyrosine
0.67
0.13
0.20
38+116 (67%) vs. 230
2.81
1.6
Histidine
0.66
0.15
0.19
43+86 (66%) vs. 195
2.45
1.3
Glycine
0.51
0.13
0.36
47 (63%) vs. 75
1.18
0.0
Tryptophan
0.49
0.07
0.44
37+199 (93%) vs. 255
4.11
2.9
Alanine
0.48
0.17
0.35
86 (75%) vs. 115
2.15
1.0
Methionine
0.44
0.36
0.20
137 (74%) vs. 185
3.43
2.3
Phenylalanine
0.42
0.16
0.42
39+155 (92%) vs. 210
3.46
2.3
Leucine
0.41
0.10
0.49
164 (96%) vs. 170
4.10
2.9
Valine
0.40
0.10
0.50
135 (87%) vs. 155
3.38
2.2
Isoleucine
0.39
0.14
0.47
155 (89%) vs. 175
3.88
2.7
Cysteine
0.32
0.14
0.54
48 (36%) vs. 135
1.20
0.0
Posttranslational modifications increase protein structural diversity
General:
Proteolysis | Racemization | N-O acyl shift | N-S acyl
shift | Other enzymatic processing:
N-terminus: Acetylation | Formylation | Myristoylation | Pyroglutamate
C-terminus: Amidation | Glycosyl phosphatidylinositol (GPI)
Lysine:
Methylation | Acetylation | Hydroxylation | Ubiquitination
| SUMOylation | Desmosine
Cysteine: Disulfide bond | Prenylation | Palmitoylation
Serine/Threonine: Phosphorylation | Glycosylation
Tyrosine: Phosphorylation | Sulfonation
Asparagine: Deamidation | Glycosylation
Aspartate: Succinimide formation
Glutamate: Carboxylation
Arginine: Citrullination | Methylation
Proline: Hydroxylation
© 2006, Alpay Taralp, Sabanci University
Bonding Diversity: Factors Determining Protein Structure & Stability
Physico-chemical properties of the amino acid side chains determine the
folded conformation
Evidence shows that the amino acid sequence of most proteins
contains all the information to arrive at the folded conformation.
Assume each amino acid adopts 2 conformations in a 250-unit chain –
We obtain 2250 ≈ 1075 conformations.
Steric constraints reduce the number, however, a very large number
of conformations is still possible.
The main factors, which cause a long polypeptide chain to fold into stable
conformation are:
Hydrophobic interactions among amino acid side-chains
Hydrogen bonding
Ionic interactions
Dipolar-dipolar interactions and hydrophilic interactions, dipolar
interactions, quadrupolar interactions
© 2006, Alpay Taralp, Sabanci University
Diversity of Protein Structural Elements: Basic Structural Hiearchy
1. Primary structure: The exact specification of atomic composition and the
chemical bonds connecting those atoms, including stereochemistry. (i.e.,
L-amino acid sequence, disulphide bridges, other postsynthetic
modifications, e.g., insulin A & B chains; chymotrypsin A, B & C chains)
2. Secondary structure: Regular arrangment of the backbone polypeptide
without reference to side-chain types or conformation. The secondary
structure is usually held by H-bonds (e.g.,  helix, b sheets, random coils)
3. Tertiary structure: 3-D arrangement of polypeptide backbone and amino
acid side-chains (e.g., lysozyme). Domain structure: compactly folded units
4. Quaternary structure: Noncovalent association of folded protein subunits
(e.g., haemoglobin)
>> Most enzymes: Globular shape, with hydrophobic interior & hydrophilic
exterior
So are protein physical traits diverse?
Compare keratin versus collagen versus albumin (all from the same 20
amino acid types)
© 2006, Alpay Taralp, Sabanci University
How do we draw protein 3-D structure?
Space filling, stick/skeletal (backbone only, sometimes labeled)
and ribbon/ schematic models:
Show  helices (coils), b strands (arrows) & random structure
Note: Proteins are made not only using amino acid components – you
must also consider water, metal ions, carbohydrates, lipids, porphorin
rings, cofactors, etc.
© 2006, Alpay Taralp, Sabanci University
Diversity of protein function
Q: What is protein function?
A: Function describes a signal transduction
a. chemical-mechanical; muscles;
b. chemical-chemical; metabolism;
c. chemical-electrical; nerve transmissions;
d. photochemical; vision & photosynthesis;
e. transport; active & passive transport;
f. defense - antibodies & blood clotting
© 2006, Alpay Taralp, Sabanci University
Classes of Protein According to Function
1.
Enzymatic proteins: Proteinases, lipases, epimerases, kinases,
polymerases...Proteins, which transduce chemical to chemical signals
Note - Proteins are not just enzymes – antibodies, connective tissue
(collagen), fluid media, transportation vehicles (Haemoglobin, serum
albumin), buffers (serum albumin), signal transducers (rhodopsin), etc.
2. Cytoskeleton – Actin (muscle), Tubulin (cell motility), Intermediate
filaments (mechanical protection near membranes and cells subjected
to stresses), Spectrin (cytoskeletal protein, particularly found in
erythrocytes)
3. Human Plasma – Albumin (osmotic regulation, buffering, transport), Globulins (transport),b-Globulins (iron transport {transferrin},
histocompatibility antigen {b2-Microglobulin}), -Globulins Antibodies,
Fibrinogen (proteolised by thrombin to form fibrin clot), Complement A
(11 different protein types working to complement the immune system)
4. Extracellular Matrix – Glycosaminoglycans (hydrated gels),
Proteoglycans (long glycosaminoglycans linked to a core protein),
Collagen (extracellular matrix; Type I-III tissue supporting fibrils, Type
IV laminar network), Elastin (random coil protein gives elasticity to
tissues), Fibronectin (cell adhesion), Integrin (integral membrane
proteins, also adhesion of cells to extracellular matrix)
© 2006, Alpay Taralp, Sabanci University
5. Digestive Enzymes of Digestive Tract – Amylase (starch to
disaccharides), Pepsin, Trypsin, Chymotrypsin (proteins to large
peptides), Peptidases (large peptides to small peptides; small peptides to
amino acids), Lipases (lipids to fatty acids and glycerol), Ribonuclease
(RNA into oligonucleotides), Disaccharidases (disaccharides to
monosaccharides)
6. Cytosol Proteins (300-1000 types) – Synthesis of most small molecules,
proteins, carbohydrates & lipids of cell
7. Nuclear Proteins – Histones (5, complex to DNA to make
chromosomes), Nucleic Acid polymerising enzymes (5-10, used in DNA
and RNA synthesis)
8. Mitochondrial & Chloroplast Proteins (300-1000) – Energy production
from metabolites or light
9. Endoplasmic Reticulum & Golgi Apparatus Proteins (50-200) Protein modification, oligosaccharide and lipid synthesis
10. Lysosome & Peroxisome Proteins (300-1000) – Degradation
processes of undesirable compounds
11. Plasma Membrane Proteins (100-500) – Transport across
membranes, transmission of important metabolic signals across plasma
membrane
© 2006, Alpay Taralp, Sabanci University
Diversity of protein physico-chemical traits:
>> Diversity among proteins is high but not “random”
>> Structure/construction and function are related
>> Some 1˚, 2˚ & 3˚ features are retained among proteins of similar function
•
•
•
•
•
•
Global shape and morphology: Round, tight, loose, fibrous, skinny,
crystalline
Local function-related structures: Active site, receptor site, allosteric
regions, catalytic residues
Solubility: Highly variable
pI: Highly variable
pH stability: Highly variable
Tolerance to other environmental factors: Highly variable
Understanding protein structure, protein function, and their
relationships are the central problems of protein science.
The rules that govern structure-function relationships are simple
Nature is presumed to provide simple solutions.
The challenge is to ask the right questions.
© 2006, Alpay Taralp, Sabanci University
What is protein chemistry?
Classic emphasis
Area of science related to:
Current emphasis
1. Obtaining/purifying protein,
2. Investigating protein structure & function, and
3. Controlling and engineering proteins
Protein chemistry contributes to the following subject areas:
1. Biochemistry, Biotechniques & Bioengineering
2. Analytical Chemistry and Spectroscopy
3. Surface and Colloid Science
4. Clinical Chemistry
5. Polymer Science
6. Medicinal and Pharmaceutical Chemistry
7. Organic Chemistry
© 2006, Alpay Taralp, Sabanci University
Why is protein chemistry highly
interdisciplinary? Protein chemistry has
developed together with analytical methods such
as sequencing, X–ray, NMR structure determination
and site–directed mutagenesis.
Protein chemistry is useful to whom?
Researchers, professionals and students in various
areas of specialization:
Protein chemists, molecular biologists, materials
scientists, enzymologists, clinicians, analytical
chemists, biophysicists and industrial scientists
© 2006, Alpay Taralp, Sabanci University
E.g.: Protein chemists help X-ray crystallographers & genetic engineers:
Protein chemist
•Purifies 1g protein
•Chemically modifies to aid crystallization
or to form heavy atom derivatives,
which aid the phase problem
X-ray crystallographer
•Attempts crystallization
•Obtains diffraction patterns
•Uses heavy atom
derivatives to solve
structures at 3-4Ǻ
Protein chemist
•Purifies 1mg protein
•Sequences peptides
•Compares peptides & sequence codes
•Probes posttrans processing by FabMS
•Prepares antibodies
•Develops protocols to purify
•Compares properties of wild-type & mutant
Genetic engineer
•Synthesizes oligonucleotides
•Screens the gene-bank
•Sequences DNA of insects
•Constructs expression vector
•Screens using western blots
© 2006, Alpay Taralp, Sabanci University
a S-F study ?
One of the most common and often ambitious experiments in protein
chemistry is the structure-function study
I.e., How does structure perturb function? How does function define structure?
e.g. Consider the pKa of active-site thiols in cysteine proteases
Structure-function experiments:
 probe the interdependence of structure & function in proteins;
 generally reflect elements of both structure & function:
Pure S study
continuum
Pure F study
Examples along this continuum:
1. One end – X-ray; Emphasizes analysis of structure
2. Middle ground - pH titration of protein groups, showing hysteresis; Reflects
elements of structure and function substantially
3. 2nd end – Bioassay; Weighted toward functional assessment
© 2006, Alpay Taralp, Sabanci University
How do S-F studies work? How would you learn
about a system that you cannot see? You interact
with the system & note the consequences.
stationary
glacier
happy
furry animal
+ achorn
moving
glacier
REGION OF INTERACTION
Interaction 2
Interaction 1
panicked
furry animal
+achorn
If you walk into an icicle, your “initial
state” becomes altered. Your “final
state” indicates something sharp.
Thus, any change in you during the
interaction can probe structure.
Structure-function studies use:
physical measurements (usually spectroscopic)
and/or chemical protocols (usually covalent
modification)
Physical methods:Generally nonintrusive,
require more protein, performed in water or
water-free state.
Chemical methods: Generally intrusive, may
be destructive. Potentially very sensitive,
performed in water, organic solvent or dry
state.
Some physical methods to assess structure & function
Diffraction: X-ray, neutron diffraction
Spectroscopies: Infrared, ultraviolet, Raman, optical rotary dispersion,
circular dichroism, NMR, esr (principle is to infer structure by perturbing
light)
Thermal analysis: Microcalorimetry
Spectrometry: Mass analysis
In silicio: Computer modeling
Other: Electrophoresis, hydrodynamic techniques, chromatographies
Typical outputs:
Composition and secondary structure, quantification, folding energies
(spectroscopies)
Identifying/purifying biological materials by exploiting adsorption,
isoelectric point, size/mass, affinity, etc. (chromatography &
electrophoresis)
Unfolding enthalpies of protein (microcalorimetry)
3-D "Static" structure (X-ray, neutron diffraction)
3-D dynamic structure, kinetic folding, association constants, etc.
(NMR)
Local environment of coordinated metal ions (Mossbauer spec.)
© 2006, Alpay Taralp, Sabanci University
Some chemical methods to assess structure & function
Titration studies: nature & number of ionizable groups.
Proteolysis in vitro: Limited proteolysis to elucidate the structural
motifs of protein.
Kinetic studies: Applied to any protein, but mainly enzymes.
Classic chemical modification: Used to identify important residues.
E.g., acetylation of chymotrypsinogen vs. chymotrypsin showed the role
of Ile16.
Competitive labeling: Very sensitive and powerful. Reports on
individual residue pKa values, structural information such as
accessibility of groups, and stereoelectronic perturbations of a group.
E.g., the surface reactivities and pKa values of the 12S subunit of a
native 50-protein ribosome complex was characterized.
Site-directed mutagenesis: Reports on the role of specific groups. All
groups can be investigated. SDM is complementary to chemical
modification. Using SDM, the role of active site groups of barnase on
stability and catalysis were quantified.
© 2006, Alpay Taralp, Sabanci University
continuum
Pure S study
Pure F study
In a typical study of a poorly characterized protein...
1. Physical & chemical methods to purify protein and to
analyze protein structure (some early examples):












Dialysis and gel filtration, column chromatography of proteins
Zone electrophoresis of proteins
Estimation of protein and amino acid content
Paper chromatography of amino acids and peptides
High-V paper electrophoresis of amino acids and peptides
Ion-exchange chromatography of amino acids and peptides
Disulphide bond mapping
Urea unfolding and stability tests
Selective cleavage of peptide chains
N-terminal sequence determination
C-terminal sequence determination
X-ray and later CD and NMR structures (with/without incipients)
2. Physical & chemical methods to analyze protein function
 Bioassays (enzyme kinetics, receptor-hormone, protein adsorption, cell
adhesion to protein layers)
 Comparative studies
give insight to the S-F relationship!
© 2006, Alpay Taralp, Sabanci University
A Review of Protein StructureFunction at Play: Enzyme
Strategies to Accelerate Rates
© 2006, Alpay Taralp, Sabanci University
An enzyme will not “reduce” the activation energy of a
pathway! Like all catalysts, an enzyme will permit the
reactants to follow an alternate, low–energy pathway.
CO → CO2
2CO + O2
The alternative pathway reflects a
new mechanism. Here, it proceeds
via 2 or more intermediate steps.
2CO2
overly simplified
Enzymes use similar tricks as nonenzymatic catalysts: E.g., bases, acids,
metal surfaces, etc., PLUS some extra
tricks, which are unique to its structure
more correct
Enzymes in the Protein Family: Properties
1. Monomeric or oligomeric or exist as part of a multienzyme
complex
2. Often require non-protein components (co-factors) for
catalytic activity – activator eg. metal ion, co-enzyme,
prosthetic group
3. Efficient catalysts
4. High Specificity
5. High Stereospecificity
6. Very sensitive to pH, temperature, dielectric (salts, solvent)
Industrial Uses of Enzymes
Textile Industry – Cellulase for cotton
Detergent Industry – Lipases and Carbohydrases for stains
Food Industry – Isomerase of glucose to fructose; lactase for
lactose intolerant people
Organic Synthesis – Penicillin acylase; amino acid synthesis
1a. Oxidoreductases (all redox reactions) eg. Alcohol to aldehyde – catalysed by NAD
oxidoreductase, aka alcohol dehydrogenase (plus NAD+ cofactor NADH)
1b. Transferases (transfer of methyl groups, glycosyl groups, phosphate groups, etc.)
eg. creatine to phosphocreatine – catalysed by creatine phosphotransferase aka
creatine kinase (plus ATP ADP)
1c. Hydrolases (hydrolytic cleavage of ester, amide and glycoside bonds by insertion of
water) eg. glucose-6-phosphate to glucose plus phosphate – catalysed by glucose-6phosphate phosphohydrolase, aka glucose-6-phosphatase
1d. Lyases (cleavage of bonds by mechanisms other than hydrolysis or oxidation;
carbon-carbon lyases, carbon-oxygen lyases, carbon-sulfur lyases) eg. L-histidine to
histamine plus carbon dioxide – catalysed by histidine decarboxylase
1e. Isomerases (racemizations, epimerizations, cis-trans isomerization) eg. D-ribulose5-phosphate to D-xylulose-5-phosphate – catalysed by D-ribulose-5-phosphate 3epimerase aka phosphoribuloepimerase
1f. Ligases (condensation of two different molecules at a new C-O or C-S bond, but
coupled to the breaking of ATP) eg. L-tyrosine plus tRNA plus ATP to give L-tyrosyl-tRNA
plus pyrophosphate – catalysed by L-tyrosyl-tRNA ligase aka lyrosyl-tRNA synthetase
© 2006, Alpay Taralp, Sabanci University
Mechanism & Strategies of Rate Acceleration
in Enzymes
General questions
 1) Why are enzymes such efficient catalysts?
 2) Which factors typically affect enzyme performance?
 Binding: Unproductive binding, competing substrates,
competing products, competitive inhibition, uncompetitive
inhibition, and noncompetitive inhibition
 Temperature
 Ionic strength, pH value and other environmental factors
 Local diffusion and convection
 3) Why have proteins been selected as catalysts in
biological systems?
 4) How large do enzymes have to be?
© 2006, Alpay Taralp, Sabanci University
Quantifying enzyme rates
Means of Quantification: Measure a change of S→P over time, many techniques
 Q: Why do we study enzyme activity?
 A: Enzyme kinetics probes protein structure and function in
general.
 Enzymes are proteins evolved with a natural marker of structure &
function.
 Q: What are some parameters to characterize
enzymes?
 A: Enzyme Units (historically)
 EU/mg protein (specific activity)
 Ks (Binding constant)
 KM (Michaelis constant)
 kcat (turnover number/catalytic constant)
 kcat/KM (specificity constant, or pH activity for kcat/KM versus pH)
 Ki (inhibition constant: competative, uncompetative, noncompetative)
© 2006, Alpay Taralp, Sabanci University
Rate measurements: Rate of formation of product or removal
of reactant as amount/time e.g., M/s, mole/s, vol/s, g/ml/s, etc.
Q: What do we call these measurements?
A: Initial rates! Acquire data within a
few minutes & within 1-5 mole% S
conversion.
Q: Why measure initial rates?
Forward rate, S → P, has no interference:
1. No product inhibition is possible;
2. No reverse reaction is possible;
3. Enzyme instability is less of a concern; and
4. Be safe - Enzyme reaction models are more
complex than ordinary kinetics: Invite errors when
extrapolating non-initial rate data
Let us examine how the above theory has
originated...
Try to measure
these slopes!
Historically
 Early studies (1895-1913) on the rates of the enzyme-catalyzed
reactions gave the following observations:
 1. At constant substrate concentration, the rate of reaction was directly
proportional to the enzyme concentration.
 2. At constant enzyme concentration:
 a. The reaction rate was independent of substrate concentration.
 b. The reaction rate was directly proportional to the substrate concentration.
 c. The reaction rate was fractional with respect to substrate concentration,
with a value between zero and one.
 In 1913, Michaelis & Menten proposed a scheme to account for
the above observations:
 Enzyme only acts upon bound substrate, i.e., E & S must initially form a
complex, held together by physical forces.
kcat
E+P
ES
E+S
 Assumptions:
KS
 E and S are equilibrated with ES, i.e., kcat << k-1
 Breakdown of ES is 1st order so rate  [ES] i.e. rate = kcat[ES]
 Rate of reverse reaction is zero
So rate = (kcat[E]o[S]o)/(Ks+[S]o)
© 2006, Alpay Taralp, Sabanci University
E+S
as k2 << k-1
KS
ES
kcat
E+P
rate = (kcat[E]o[S]o)/(Ks+[S]o)
E+S
k1
k-1
ES
k2
E+P
rate = (k2[E]o[S]o)/(KM+[S]o)
 Briggs and Haldane revised the mechanism
 They assumed that k2 was significant in comparison to k-1
(not an equilibrium, rather a steady-state).
 They set d[ES]/dt = zero to obtain a rate formula.
 The “new” M-M equation has the same form as the
original! Why? Equilibrium is a special case of the steady
state treatment, k2 << k-1.
 How does KM vary amongst the two models? KM is either
(k-1+k2)/k1 or KM ≈ KS = k-1/k1 (in the original M-M model).
© 2006, Alpay Taralp, Sabanci University
Q: What are enzyme assays & how are they performed?
SP
rate = (kcat[E]o[S]o)/(KM+[S]o)
 The Assay
 Any method that detects a change of physical property versus time:
Manometry, polarimetry, viscometry, NMR, MS, spectrophotometry,
spectrofluoromethry and pH-stat. What is one pre-condition? The
physical property should vary in proportion to S or P.
 Direct assays
 Alcohol dehydrogenase can be monitored as a function of NADH
formation. NADH is strongly absorbent at 340nm. Is a buffer used?
 Hydrolases can be monitored as a function of proton formation (standard
ester cleavage). Is a buffer used here?
 Coupled Assays
 If S & P are similar they cannot be directly used to assay. To get around
this problem, a more distinguishable end product is made.
 Target: With alanine aminotransferase; alanine + -ketoglutarate →
pyruvate + glutamate. Using pyruvate dehydrogenase; pyruvate + NADH
→ lactate + NAD+ (NAD+ absorbs at 260nm). The coupled reaction
should be faster than the principle reaction. WHY??
© 2006, Alpay Taralp, Sabanci University
Sampling Assays
S or P is withdrawn at specific time intervals & quantified, e.g., by
colorimetry or radioisotopy.
Experimental Target of a M-M assay
To measure 3 parameters: KM, kcat & kcat/KM. Do these carry a
physical meaning?
Q: How do we carry out a typical M-M experiment?
A: Measure the initial rates as follows:
With substrate concentration at least 200-500x greater than total
enzyme concentration , measure KM& kcat directly. Carry out these
measurements at 3-4 different pH values.
Measure the specificity (kcat/KM) directly at many pH values, using
0.1pH unit intervals (construct a pH activity curve); In choosing
your parameters, S must be at least 20x less than KM. Why? What
is the significance of a pH activity curve?
Repeat any of the above experiments in the presence of
inhibitors, different S, activators, different environments, etc.
Q: How does your experimental scenario compare to the true
situation in biological systems? Is there a biological relevance? Why do
we conduct experiments in this way?
rate = (kcat[E]o[S]o)/(KM+[S]o)
Initial rate (mM/s)
Hanes-Wolf plot
Michaelis-Menten kinetics
= KM/(kcat[E]o)
Conc of S (mM)
A closer look at kinetic scenarios: Probing ionizable groups,
which are important for binding and/or catalysis?
(6)
=0
Sample math treatment
for 3 (apparent)
ionizable groups that are
important for binding
and/or catalysis
The pH activity profiles of cathepsin B. The substrates are acetyl-Arg-Arg-ArgAMC (+), acetyl-Val-Arg-Arg-AMC (◊) and benzyloxycarbonyl-Arg-Arg-AMC (―).
140000
120000
100000
80000
kcat/Km
Real
example!
60000
40000
20000
0
3
4
5
6
pH
7
8
9
Thermokinetic background related to protein analysis
Thermodynamics: DG, DH, DS, equilibrium constant Keq
Kinetics: DG≠, DH≠, DS≠ , kinetic rate constant k, kinetic rate theories
Origin: Position of G, H & S changes as system proceeds along reaction coordinate
Plan: To discuss the interrelation of these parameters and to focus on DG≠ and DG
© 2006, Alpay Taralp, Sabanci University
Put away your
weapons of mass
destruction...
Please delinate the relative importance of thermodynamic and
kinetic contributions in the following scenarios 
1. The right reaction releases energy faster than the left reaction.
Q: Which videoclip shows the more exothermic reaction?
A: Inconclusive! We cannot compare the molar enthalpy change
from the videos.
2. True or false? All exothermic reactions are thermodynamically
spontaneous and all endothermic reactions are thermodynamically
non-spontaneous.
A: False!
3. True or false? All thermodynamically spontaneous reactions yield
a reaction & all thermodynamically non-spontaneous reactions fail.
A: False!
4. The thermite reaction is highly exothermic, DH <<<0, the
entropy change, DS, is relatively unimportant, and the Gibbs
energy change is highly negative, DG <<<0. The reaction is
thermodynamically spontaneous.
Q: Why must you add a fuse to start the reaction?
5. The process H2O(s)→H2O(l) is highly endothermic (DH>>0)
Below is the evidence. Explain.
Time = 0min
Time = 60min
NI3.NH3(crystal) → NH3(g) + ½ N2(g) + 3/2I2(g)
6. The reactant, nitrogen triiodide-NH3, sits at a high Gibbs energy
level. Its products rest at a much lower energy state.
You must apply a physical shock before Nitrogen triiodide-NH3
explodes. Why?
7. Liquid nitrogen evaporates. The process is thermodynamically
spontaneous, endothermic & proceeds quickly.
Q: How might you explain these comments?
N2(l) → N2(g)
8. The dissolution of ammonium sulfate in water is endothermic and
readily proceeds under ambient conditions. Explain.
(NH4)2SO4 + bulk H2Os → 2NH4+(aq) + SO42-(aq) + a few less bulk H2Os
Why all the confusion??
Reason 1: Many terms and reactivity models
Reason 2: Misleading terms
Reason 3: Separate GS & TS concepts in chemical
processes
At equilibrium, S sys + Ssur is max.
Gsys
H
U
Asys
TS U TS
Let us progress
until we
arrive at the
common
model to
understand
proteins...
PV
potential Emic
intermol.
inter.
kinetic Emic
transla intramol. inter.
rot vibr
Early measurements of DU examined the link between enthalpy
changes (DH ≈ differences of bond energies) and reactivity
A
Hinitial
H
B
Exothermic
Endothermic
DH < 0
B
Hfinal
Reaction coordinate
DH > 0
A
Reaction coordinate
Why shouldn’t you predict reactivity using DH?
A: DH reports on the initial & final Ground States (GS)
but not on the pathway (mechanism)
(There are other reasons too)
Collision model: A kinetic view. Consider a potential barrier, Ea,
between A & B. Rate const is kA→B = Ae-Ea/RT. (Later, A = Zr)
preexponential
steric
Reactants collide with
speed & good
orientation.
In non-gases:
 DPotE ≈ DU, as
(PotEf - PotEi) ≈ Uf - Ui
 DU ≈ DH, as
DH = DU + D(PV)←very small
A
DU
B
What are some
disadvantages of:
 the collision model?
 using Ea to predict
reactivity?
Gibbs energy change: A way to explicitly incorporate
entropy, S, to account for solvent effects, etc.
At equilibrium, S sys + Ssur is max.
DH-TDSsys = DGsys
Gsys
H
most reactions
U
TS U TS
PV
DG < 0
What is misleading
by the term
spontaneous?
Asys
bomb calorimeter
DG > 0
Spontaneity says nothing about energy barriers or chemical
rates
DG≠
Both processes are
thermodynamically
spontaneous
One is kinetically permitted,
giving an observable rate,
Gibbs
Energy
DG
and one is kinetically prohibited
by a high energy barrier
Both processes are
thermodynamically
non-spontaneous
Products
Reactants
Reaction Coordinate
DG≠
One is kinetically permitted,
giving an observable rate,
and one is kinetically prohibited
by a high energy barrier, so we
have 100% reactants
DG
Products
Reactants
Reaction Coordinate
Transition State Theory: A kinetic element completes the Gibbs
Reactant (A) proceeds through a high-energy transition state
view.
or activated complex to become product (B).
DG‡
State A
Not a state
function
G
DG‡
State
function
DG
State A
State B
Changes of any state function
are independent of path
State B
Reaction Coordinate
Problems with
TS theory?
Quantum tunneling kinetic view: The e- probability distribution
of every particle is derived from a wave function
H
5A
-
O
5A
H
O
What is the weakness of
predicting reactivity
using only quantum
tunneling?
The rate of proton transfer
often has a significant
tunneling component
Classic kinetic
behavior
Gibbs
Energy
R,
eg. +H
Reactants
Tunneling (a 5A wavelength decays exponentially
as it penetrates the barrier. If the barrier is
not too long, R can reach the product side
P of the hill without completely decaying away (i.e.,
emerges on the product side with a non-zero
probability density)
Products
Reaction Coordinate
To summarize: In protein systems, we assess
thermodynamic & kinetic behavior in terms of G & TS
theory (less use of Zr or tunneling arguments)
DG, DH, DS
DGA→B = DHA→B – TDSA→B
kA→B = (kBT/h) x e-DG‡/RT
Keq =
[B]b/[A]a
=
e-DG/RT
DG≠, DH≠, DS≠
The above terms are related to large populations
Cannot use TS theory to calculate the activity of “one” molecule or
small groups of molecules, such as membrane proteins
Note: Reactions do tunnel, collision theory could apply
Let us examine a typical enzyme reaction...
Enzymes lower DG‡ (i.e., G‡ - GGS) in
comparison to the uncatalyzed reaction
Uncatalyzed reaction
G
Enzyme reaction
G
ES≠
S
X
+
P
Reaction coordinate
Reaction coordinate
E+S
ES
=
ES
E+P
Overall rxn is diffusion-controlled or rxn-controlled
We shall simplify the notation even more...
 Microscopic steps may be grouped into
 Physical binding (1st step; E + S → ES), and
 Chemical catalysis (2nd step; ES → ES‡ → E + P)
E+S
k1
ES
k2
E+P
k-1
G
Two models:
TS lowering & GS
elevation
Reaction Coordinate
© 2006, Alpay Taralp, Sabanci University
A closer look at changing the position of G
G
Q: How might you predict the free
energy of activation, DG‡?
Ggs
activation
energy of
forward
process
Gibbs
Energy
Answer: Assess the enthalpic &
entropic differences between:
1. reactant (ES at ground state) &
k1
E+S
ES
k2
E+P
k-1
Reactants
2: the activated complex (ES≠, at
the transition state position).
Products
Reaction Coordinate
Dissect DG‡ into enthalpic (DH‡) and entropic (DS‡) components:
k = (kBT/h) x e-DG‡/RT can be written as k = (kBT/h) x eDS‡/R x e-DH‡/RT
where DS‡ is the entropy of activation, Stransition state – Sground state and
DH‡ is the enthalpy of activation, Htransition state – Hground state.
H
H
H
H
activation
enthalpy of
two forward
processes
The enthalpy of activation , DH‡,
is always positive because bonds
are being broken.
A
S
activation
entropy of
two forward
processes
S
S
A
B
S
Reaction Coordinate
Reaction Coordinate
The entropy of activation, DS‡, may or may not be
favorable. Can you think of some examples?
Both parameters contribute to rate according to
k = (kBT/h) x eDS‡/R x e-DH‡/RT
Q: How might substrate-surrounding interactions affect the
position of H?
(Hint: In solutions & solids, DH ≈ DU)
Oil
Water
Enthalpy,
H
Nonpolar solvent
Polar solvent
Reaction Coordinate
Q: If you ignore any entropic contribution, how might a change
of H affect the Gibbs free energy position in solutions & solids?
Oil
Water
Gibbs Energy,
G
Nonpolar solvent
Polar solvent
Reaction Coordinate
Q: What happens if you increase the chemical potential (i.e.,
the potential to do work) of a reactant?
Increasing the
concentration of
reactant
Gibbs
Energy
Answer: Reaction is more
spontaneous; equilibrium
is even closer to the
product side; transition
state is reached earlier;
activation energy is
smaller; forward rate is
greater
Equilibrium
position, before &
after
Products
Reactants
Reaction Coordinate
Q: Which principles do enzymes exploit to lower the
position of the TS (& how)?
 1. General acid catalysis, general base catalysis,
electrostatic catalysis and electrophilic catalysis.
 All modes could stabilise charge accumulation in proceeding
from ground state, GS, to transition state, TS.
Hydroxide
Enzyme
© 2006, Alpay Taralp, Sabanci University
2. Covalent or nucleophilic catalysis.
A covalent activated intermediate is formed, e.g., a ping-pong
mechanism. The high-energy mechanism is broken into energetically
less-demanding steps.
G
O
O
H2O
OH
N
H
H
H
-
Enz
O
O
O
H2O
O
N
H
N
H
O
Enz
Enz
OH
-
O
N
H
3. Neighboring charges, dipole moments
& hydrophobic/dielectric considerations.
The enzyme environment enhances the
reactivity of nucleophiles such as serine
hydroxyl groups, cysteine thiol groups, etc.
His
HN
pKa = 3
Cys
+ NH
S
-
pKa = 9
Cys
(aq)
SH
-
CH3CH2O + ICH2CH3
LSDS≠
O
versus
<<
RSDS≠ I
4. Pre-reduction of ground-state entropies.
The basis of this strategy is to minimize the ground
state freedom of the ES complex during the chemical
transformation phase of a reaction. In this way, the
ascent to the TS will not require a major loss of freedom.
Strategy 1. Orbital steering.
O
Enz
N
H
A
(aq)
O
versus
A
O
N
A
H
N
H
Strategy 2. Decrease the number of reaction
participants in the chemical transformation phase.
≠
+
versus
≠
+
Enz
Enz
5. Formation of low-barrier H-bonds.
A normal H-bond in the GS may become a low-barier
H-bond in the TS if the pKa value of the enzyme group &
the activated complex (as ES≠) are matched.
1 normal H-bond ≈1-5 kcal/mole
1 low-barrier H-bond ≈ 25-40kcal/mole
TS
pKa = 10
GS
pKa = 16
O H
-
O
H
Enz
NH
+

Od
H N
H3C
pKa = 10
S
H3C
S

H dO
Enz
6. Binding energy considerations.
Enzyme binding groups interact non-covalently with
substrate at all points along the reaction coordinate. The
energy term H is variable along the reaction coordinate!
E.g. 1, GS shape of S perfectly matches enzyme site
G
GS
TS??
E+S
+
S
Enz
10 good
contacts
Conclusion: Groundstate binding shouldn’t
be “extremely specific”,
as is often assumed
ES
G
GS
Shape of S transforms along
the reaction coordinate
TS
=
ES
+
S
Enz
2 good
contacts
10 good
contacts
E+S
E+P
ES
E.g. 2, TS shape of S is much more complementary to
enzyme than GS shape of S (for enzymes that behave
according to the TS stabilization model of catalysis).
In a well-evolved enzyme-substrate interaction, we see an
increase of binding energy stabilization in proceeding to the TS
+
S
Enz
2 good
contacts
3 good
contacts
Every “good” interaction
lowers the position of H, etc.
5 good
contacts
10 good
contacts
8
G
6
=
ES
TS reached
3
6 good
contacts
8 good
contacts
10 good
contacts
E+S
2
ES
E+P
Q: Can you see evidence of binding energy
participation in the TS of amide bond hydrolysis?
Hint: Look at the changes of KM & kcat
© 2006, Alpay Taralp, Sabanci University
=
S (Uncatalyzed energy profile)
G
Summary slide 
rate-determining
transition
E+S
physical
binding
=
ES
ES=
ES
chemical
transformations
At equilibrium, S sys + Ssur is max.
contacts
Gsys
=
ES
(Enzyme-catalyzed energy
profile including binding
energy contribution)
4
H
U
E+S
ES
2
physical
dissociation
(Hypothetical enzyme-catalyzed
energy profile when binding energy
is not considered, i.e., profile is
analogous to non-enzymatic
catalysis)
10 good
8
E+P
EP
EP
Reaction Coordinate
E+P
Asys
TS U TS
PV
potential Emic
intermol.
inter.
kinetic Emic
transla intramol. inter.
rot vibr
va = Vmax = kacat[E] o
(a)
A
va = Vmax /2
Case 1 (not shown here): ES has
very strong GS binding (shape
complementarity is exceptionally
good in the ground state). Examples:
Hormones
Case 2: ES has poor GS binding
and strong TS binding. Examples
include carbonic anhydrase,
acetylcholine esterase and catalase.
Case 3: Modest GS binding and
modest TS binding. Examples –
Metabolic enzymes
B
Increasing
reaction
rate (v)
observed
C
D
[S] = Kam
b
d
a ; va = (kacat/Kam)[S] o[E]
c
ES binding energies can be
grouped into three cases:
[S]o >> Km
[S]o << Km
Increasing [Substrate]
(c)
Small [S]
Increasing
Gibbs energy
of free
substrate
& substrate
in enzyme
complexes
+
+
ESc,d
+
+
ES a,b
( kcat) a,b
Km
S
ESa,c
ESb,d
Large [S]
(b)
+
ES +
C,D
+
ES +
A,B
->P
kSuncat
( kcat ) A,B
Km
S
ES
A,C
B,D
Km
ESB,D
stabilization before binding
energy consideration
kB
cat
P
P
Product
Substrate
Substrate
Product
forward rate const = k cat
forward rate const = k cat/Km
Reaction Coordinate Axes
© 2006, Alpay Taralp, Sabanci University
Protein engineering to elucidate and
improve stability
© 2006, Alpay Taralp, Sabanci University
Protein engineering has been used to investigate structure-activity &
molecular recognition relationships to to make better protein products
Q: Why does Mankind wish to use proteins?
 Proteins accelerate chemical reactions
 Proteins form commercial products & improve other product properties
 Proteins enable novel processes
Some typical industrial applications:
 Bioreactors
 Textile treatment
Improved Enzymes
 Medicinal and organic syntheses
 Protein drugs & drug delivery
 Biosensors
 Bioremediation
Structure-Activity
Relationships
 Food preparation industries
Redesigned Antibodies
'synthesis'
Improved Proteins
'analysis'
Molecular Recognition
 Problems? Industrial constraints are often too demanding for native proteins.
 Consequences: Poor biological activity, short lifespan, limited reaction parameters, etc.
Q: What can we accomplish by using protein engineering?
 improve existing pharmaceutical proteins
 create superior high-value proteins with improved half-life
 create new proteins and pioneer new therapies
 improve desirable biological activities
 alter receptor specificity and binding activity
 reduce harmful side-effects and toxicities.
© 2006, Alpay Taralp, Sabanci University
The current focus of protein engineering: Formulating
broad-scope protein preparations, which are:
 Cheaper
 More stable
 More catalytic
 Longer-lived
 More easily stored
& transported
 More active at pH &
temperature extremes
Locating/purifying thermophiles, etc.
Native
Low-tech
chemical strategies
The current focus
is genetic
manipulation
Genetic
manipulation
focus
PROTEIN ENGINEERING
© 2006, Alpay Taralp, Sabanci University
Interacting
residues are
observed
Barnase: Superimposed
Xray/NMR, schematic
and ribbon sketch
Q: Can mutations probe the stability of the folded state?
A: All residue interactions contribute to protein stability.
By mutating 1 residue of an interacting pair, the Gibbs
contribution of that pair to protein stability is assessed.
G
E' =
E=
Eu, E' u
E'i
Ei
E'f mutant
Ef
wildtype
Reaction Coordinate
How might you measure the thermodynamic
unfolding/folding energy change of barnase?
x
G
E' =
E=
x
Fluorescence
of Trp residues
mut
Eu, E' u
E'i
Ei
x
E'f mutant
Ef
wildtype
Concentration
corresponding
x to 50% fluorescence
x quenching
x
x
xx x
wt
Concentration of denaturant
Reaction Coordinate
Folded
Trp's
inside
Unfolded
Trp fluorescence
quenched
In principle, all interactions contribute to protein stability. H2ODGUnfolding
is the free energy change (calculated), which accompanies
barnaseFolded → barnaseUnfolded in water. Here are some examples:
Deleting one H-bonding partner where
there is no access of water
Mutant
[urea]1/2
H2ODG
U
(in M)
DDGU
(kcal/mole)
wt
4.57
8.82
----
TyrPhe78
3.88
7.68
1.35
SerAla91
3.58
6.41
1.93
Introducing a H-bonding residue in a
place that contains no partner residue
Mutant
Deleting one H-bonding partner
where there is free access of water
Solvent access-
DDGU
ible area (in Ǻ2)
(kcal/mole)
ValThr10
0
2.48
DDGU
ValThr89
0
2.55
(kcal/mole)
ValThr45
43
2.44
SerAla31
-0.14
ValThr36
70
1.15
TyrPhe103
0.00
ValThr55
93
0.60
Mutant
© 2006, Alpay Taralp, Sabanci University
Destroying parts of buried or solventaccessible hydrophobic residues
Mutant
# methyl(ene)
groups < 6Ǻ
DDGU
(kcal/mole)
Destroying a solvent-exposed ionic
interaction between Asp8, Asp12 & Arg110
DDGU
IleVal55
5
0.30
IleAla55
16
1.15
ValAla10
37
3.39
AspAla8
0.89
IleAla88
55
4.01
AspAla12
0.31
LeuAla14
62
4.32
AspAla8 & AspAla12
0.80
Mutant
(kcal/mole)
Summary: Relative importance of types of interactions towards stability
H-bonds, no access to water → moderate
H-bonds, free access to water→ very small
Hydrophobic-hydrophobic interactions → very important, very abundant
S(64 mutations) → >60kcal/mole destabilization energy
© 2006, Alpay Taralp, Sabanci University
Mutation studies have validated the importance
of some interactions. Can we use site-directed
mutagenesis to engineer proteins with
enhanced stability?
Yes! Bridged mutants show resistance
to denaturants & thermal stability!
Stability of Barnase double mutants
Mutant
[Urea]
to unfold 50%
wt
DDGU
(kcal/mole)
8.8
0.0
AlaCys43
SerCys80
(–SH)
7.7
1.1
AlaCys43
SerCys80
(–S-S-)
10.0
-1.2
SerCys85
HisCys102
(–SH)
8.4
0.4
SerCys85
HisCys102
(–S-S-)
12.9
-4.1
© 2006, Alpay Taralp, Sabanci University
SDM may be aided by evolutionary clues left by Nature
 With respect to Barnase, Binase has lost one amino acid
(Gln2 → D) and has 17 different residues. The structure
of Binase is slightly more stable than Barnase.
 Hypothesis: Evolution may have selected some of the
17 amino acids because they promoted stability. If these
amino acids are mutated into Barnase, the engineered
Barnase may have higher stability...
© 2006, Alpay Taralp, Sabanci University
 Strategy to improve the stability of Barnase:
 One by one, re-engineer the primary structure of
Barnase using each of the 17 residues of Binase
 Measure the conformational stability of the mutant
 Design a “super” stable mutant using the information.
Mutation
50%Unfold
DDGu
(For comparison, wt barnase
unfolds %50 in 8.8M urea)
Grand Results:
© 2006, Alpay Taralp, Sabanci University
Take-home message
 Effects of these individual mutations are remarkably
additive
 Normally cooperativity is observed
Explanation
 Binase and Barnase are slightly divergent on the
evolutionary tree
 Mutations are very conservative
Implications for any industrial enzyme such as Xylose
Isomerase
 Find a closely related thermophile
 Make individual mutations between them and determine
DDGu
 Choose the stabilizing mutations and create a multiple
mutant, stable at high temperatures
© 2006, Alpay Taralp, Sabanci University
Other examples protein engineered via genetic manipulation
have relied on the principle of directed evolution
 Directed evolution is a technique, which accelerates
evolution.
 Evolution normally takes millions of years to produce an
improvement; accelerating the mutation process yields
improvements in weeks.
 One type of directed evolution is called molecular breeding
 Desired genetic trait obtained from a two-step process:
 1. Genes are subjected to DNAShuffling, generating a diverse library
of novel sequences (one or more genes are fragmented and
recombined).
 2. “Good” gene products are selected by screening.
 The good genes are subjected to more “shuffling” & screening until
the desired property is obtained
Left image: Wild type green fluorescent
protein gene in plants
Right image: Maxygen’s DNA shuffled
green fluorescent protein gene in plants
© 2006, Alpay Taralp, Sabanci University
Protein Folding
© 2006, Alpay Taralp, Sabanci University
Folding of Barnase - Overview
 Elucidating rules, which govern the folded conformation of
proteins, is of theoretical interest and practical importance
particularly since advances in recombinant DNA methods
have enabled the design and synthesis of novel proteins.
 Although many physico-chemical approaches have been
employed, the mechanism of protein folding remains unclear.
 An approach, which combines the technique of site-directed
mutagenesis with the more classical physico-chemical
techniques, has been employed to address this problem.
 By altering specific side chains in a folded protein, it is
possible to correlate the contributions of their interactions
towards the overall stability of the protein. Thermodynamic
relationships, specifically Bronsted relations, are employed in
this treatment.
 Barnase, a relatively small 110 amino acid, monomeric
extracellular ribonuclease of Bacillus amyloquefaciens serves
as a model protein for this study.
© 2006, Alpay Taralp, Sabanci University
Protein Folding
Protein folding is a large-scale
continuation of the conformational analysis
problem
3 mutual gauche interactions
Less stable
2 mutual gauche interactions
More stable
The New Challenge!!
© 2006, Alpay Taralp, Sabanci University
Protein structure is not rigid
1. Some native structures are more
flexible and dynamic; some are tight, less
dynamic and well protected
2. We note a correlation between protein
flexibility and crystallizability
Article “What does it mean to be natively
unfolded?”
Implications for NMR and Xray analysis?
 Q: Why does a protein fold?
 A: Balance of enthalpic terms (non-covalent interactions) and
entropic terms (freedom decreases as conformation organizes)
DG = DH - TDS
Eu = unfolded enzyme
Gibbs
energy
Eı = intermediate
EU
EF = folded enzyme
Rate
Determining
Step
EI
Typically,
DGU→F = -5 to -15kcal/mole
EF
Reaction coordinate
© 2006, Alpay Taralp, Sabanci University
 Q: Please estimate the conformational possibilities
while a protein folds
 A: In a 100 amino acid chain
 8 conformations each
 up to 8100 conformations possible
 Q: Is protein folding random?
 If 1011-1013 conformations are randomly adopted per
second → requiring years to fold!
 In fact, a protein folds while associated with
 Chaperon
 Ribosome
 Alone
in msec-to-sec time scale
 A: Folding is clearly a directed process!
© 2006, Alpay Taralp, Sabanci University
 Q: How might you define the mystery of protein folding
 A: How does the amino acid sequence
 Direct folding?
 Determine the final conformation?
 Q: How might you address the problem?
 A:
 Obtain amino acid sequence (protein chemistry, DNA, molecular biology)
 Obtain 3-D structure (X-ray, NMR)
 Perturb the physico-chemical traits (Chemical modifications and site-directed
mutagenesis)
 Q: Why the interest to understand protein folding?
 A:




Predict 3-D structure of any amino acid chain
Novel enzyme design
Improved industrial processes, e.g., a better xylose isomerase → € 
Treatment of protein related diseases, e.g., prion diseases (BSE, fatal familiar
insomnia, etc.)
© 2006, Alpay Taralp, Sabanci University
In the prion class of diseases, why does a
misfolded protein lead to disaster?
G
Globular protein
non-associating
soluble
degradable
Fibrous protein
aggregating
crystallizing
insoluble
accumulating
In prion disease?
Crystal nucleation?
Normal
State
Pathological
Condition
Reaction coordinate
Today’s focus is
related to Prof.
Alan Fersht’s
work on the
folding pathway
of Barnase
R'
O
+
BH
-
O
H
B
O
H
P
-
O
O
O
P
O
O
H
B
- R'OH
O
+
HB
O
+H2O
O
O
G
RO
G
RO
Barnase: RNA → ribonucleotides
O
H
N
HN
N -1
Base 0 (guanine)
H2N
Sugar 0
N
N
P0
O
+
BH
-
O
O
P
O
H
P +1
O
Bond cleavage
N +1
O
RO
P +2
References:
N +2
Serrano, Day and Fersht (1993) J. Mol. Biol. 233, 305-312
Fersht, Matouschek and Serrano (1992) J. Mol. Biol. 224, 771-782
Serrano, Matouschek and Fersht (1992) J. Mol. Biol. 224, 805-818
© 2006, Alpay Taralp, Sabanci University
G
H
B
Q: Why is Fersht’s work interesting?
Approach uses powerful protein engineering
Novel proteins can be made, e.g., M. Smith, UBC
Improved Enzymes
Redesigned Antibodies
'synthesis'
Improved Enzymes
Structure-Activity
Relationships
'analysis'
Molecular Recognition
Data interpreted via thermodynamic treatment
Linear free energy relationships
Bronsted plots (Bronsted catalysis eqtn)
Q: Advantage of Fersht’s approach?
Relates measureable data to specific noncovalent interactions, which govern protein
structure & function
© 2006, Alpay Taralp, Sabanci University
Q: Why barnase as a model?
Advantages:
Small monomeric protein
No disulphide bridges or cis-prolines
No post-translational modifications
Excellent expression systems (wt → express;
mutants → express)
© 2006, Alpay Taralp, Sabanci University
Ribbon structure of Barnase
© 2006, Alpay Taralp, Sabanci University
Schematic
of Barnase
© 2006, Alpay Taralp, Sabanci University
Q: What was the experimental strategy?




Choose a significant non-covalent interaction
Make a subtle change to the interaction
(e.g., Ser80 → Thr80; Ser85 → Thr85)
Perform equilibrium/kinetic un/folding experiments and
compare the wt & mutant
 Rationale: All interactions contribute to protein stability - Some
form/break before the rate determining step of folding, whereas
others form/break afterwards
© 2006, Alpay Taralp, Sabanci University
X-ray is used to identify interacting residues
X-ray, NMR, CD and bioassays are used to
check correct folding of mutants
Superimposed X-ray
and NMR backbone
positions of Barnase
To recap, how might you measure the
thermodynamic unfolding/folding of barnase?
x
G
E' =
E=
x
x
Fluorescence
of Trp residues
Concentration
corresponding
x to 50% fluorescence
x quenching
x
x
Eu, E' u
E'i
Ei
E'f mutant
Ef
wildtype
xx
x
Concentration of denaturant
Reaction Coordinate
Folded
Trp's
inside
Unfolded
Trp fluorescence
quenched
Rapidly mixed
Barase solution is
spiked with acid
Shape analysis
of curve yields
kinetic unfolding
constants
Fluorescence
of Trp residues
Now, how might you
measure the kinetic
unfolding of
barnase?
Time
...and how might you Fluorescence
measure the kinetic of Trp residues
folding of barnase?
Rapidly mixed Barase
in 8M urea is diluted
10x with aqueous buffer
Shape analysis
of curve yields
kinetic folding
constants
Time
 A closer look at the consequence of a mutation
 Any measured energy change within the protein is attributed to the mutation
 You may compare (one at a time) the interactions of many neigboring
groups within the protein
 You can map interactions that do/don’t contribute to protein stability/folding
Q: What principle allows you to identify
conformational energy changes from
measurable mutation studies?
A: DGU, DGI, DGF, DG, etc. are changes
of free energy upon mutation (where DGA
is stateEAwt – stateE’Amutant).
Effect of a mutation varies along the
reaction coordinate. E.g. if you compare
wt
mutant, DG is very close to
Eu, E' u
stateEA – stateE’A
U
zero, whereas DGI, DG & DGF are larger.DG ≈ 0
U
The change of free energy upon
mutation between 2 x-positions, e.g., from
unfolded → → → → folded, is DGF-DGU.
E' =
E=
G
E'i
Ei
© 2006, Alpay Taralp, Sabanci University
DGI
DG=
E'f
DGF
Reaction Coordinate
mutant
Ef wildtype
Q: Is there a fundamental problem with the calculation of
DGF-DGU? (DGA is stateEAwt – stateE’Amutant) EU
EI
E
=I
 A: Yes! All vertical
equilibria are virtual!
DGU
E'U
DGI
E'I
DG =I
E'=I
EF
DGF
E'F
Solution: Calculate instead the difference of free energy
upon mutation, e.g., for unfolded → folded, we want
DDGF-U, so measure DGF-Uwt-DGF-Umutant (= DGF-DGU!).
© 2006, Alpay Taralp, Sabanci University
Q: What is the meaning of DDGF-U?
A: If X & Y interact and we mutate X → Z, then:
DDGF-U = GF(X...Y) + GF(X...E) + GF(Y...E) + GF(X...H2O) +
GF(Y...H2O) – G’F(Z...Y) – G’F(Z...E) – G’F(Y...E) –
G’F(Z...H2O) – G’F(Y...H2O) – G’F(E...DH2O) – GU(X...H2O) –
GU(Y...H2O) – GU(E...H2O) + G’U(Z...H2O) + G’U(Y...H2O) +
G’U(E...H2O) + GU(reorg) – GF(reorg)
Once all the terms are considered (many cancel),
we can say that DDGF-U ≈ stabilization energy!
Q: Why is this statement significant?
A: Stabilization energies probe transition states &
intermediates
© 2006, Alpay Taralp, Sabanci University
 Q: What is the advantage of DDGF-U?
 A: All horizontal equilibria are measureable via a
thermodynamic equilibrium experiment, i.e., DG = -RTlnK &
a kinetic experiment, i.e., k = (kBT/h)e(-DG/RT)
DGI-U
EU
EI
DGU
E'U
E’I
DGI
E'I
DG =I -U
E=I
DG =I
E'=I
DGF-U
EF
DGF
E'F
To compare the thermodynamics of
folding: DDGF-U = -RTln(50%urea’/50%urea)
Convention: DDG < 0 if E’F is more stable
© 2006, Alpay Taralp, Sabanci University
Bronsted catalysis equation
 Please recall logk = blogK + C
 0 < b < 1: So what does b
describe?
logk
x
x
x
x
x
x
x
b
logK
 For a “series” of 2 related compounds (e.g., wt &
mutant) you may write Dlogk = bDlogK
 Another way to write logk = blogK + C is
DG = bDGeq + D. If we blend this logic, we obtain:
b = DDG/DDGeq for wt & mutant
 We may define for each wt/mut pair the following:
fAunfol = DDGA-F/DDGU-F NOTE – f approximates b; f is not b
fAfol = DDGA-U/DDGF-U but f is equated to b when f = 0 or 1
© 2006, Alpay Taralp, Sabanci University
We begin by probing the rate
determining TS of unfolding (easier)
fAunfol = DDGA-F/DDGU-F
 Each point (x,y) describes an energy change
(DDGU-F, DDG-F) due to a mutation of barnase.
E.g., Val→Ala gives Ala:Phe
 Is there a patterned change
of stabilization energies
upon mutation of
interacting pairs?

DDG-F; DDGU-F
© 2006, Alpay Taralp, Sabanci University
Q: Let us quantify the mutant pair interactions
as barnase unfolds.
Equilibrium measurements
DDGU-F = 2.32 kcal/mole
Kinetic measurements
DDG≠-F = 2.60 kcal/mole
funfol = DDG-F/DDGU-F
= 2.60/2.32 = 1.06 ≈ 1
Therefore, the Val:Phe interaction
in the folded state was broken in the
TS of unfolding!
funfol = DDG-F/DDGU-F
© 2006, Alpay Taralp, Sabanci University
Q: Why must this kinetic data reflect the rate
determining transition state of unfolding?
A: All subsequent steps are kinetically unimportant
High [Urea]
Unfolding
For each mutation:
find DDG-F using a kinetic
experiment (urea/acid pulse) &
find DDGU-F using an
equilibrium unfolding
experiment (urea to denature
50%)
The rest is easy!
funfol = DDG-F/DDGU-F
© 2006, Alpay Taralp, Sabanci University
The state of interactions in the transition state
of Barnase with respect to the folded state
© 2006, Alpay Taralp, Sabanci University
Q: What is the general picture of the TS?
 Native-like, compact
 Some tertiary interactions lost
 Majority of 2˚ structure preserved
 Core1: weakened, esp. at Nterm 1
 Core2: completely disrupted
 Core3: fully intact
If we were to evaluate the unfolding pathway:
 1st events: 3 of 5 loops unfold, Nterm of helices
melt, core1 weakens and core2 is destroyed; the
remaining structure is disrupted later
© 2006, Alpay Taralp, Sabanci University
Probing the rate determining TS of folding and
an intermediate
Rationale:
Under refolding conditions:
EU → EI is faster than EI → EF;
Thus, your measurements can examine EI → EF
Refolding experiments
Low [Urea]
Refolding
For each mutation:
Use the appropriate kinetic
experiment (dilution of urea) &
equilibrium folding
experiment (urea) to probe the I
state and TS of folding!
© 2006, Alpay Taralp, Sabanci University
The state of interactions in the intermediate & transition
state of Barnase with respect to the unfolded state; the
last column shows the unfolding pathway for comparison
© 2006, Alpay Taralp, Sabanci University
What can be said about the folding process?
© 2006, Alpay Taralp, Sabanci University
© 2006, Alpay Taralp, Sabanci University
© 2006, Alpay Taralp, Sabanci University
Q: What can be said about folding?
 Correlation between hydrophobic burial and early events
 All early regions inteact with b sheet
 Early burial is hydrophobic, extensive and nucleated
 Late processes only interact slightly with b sheet; no
core nucleation; some hydrophillic burial is noted
General statements:
 The refolding pathway is at least partially sequential
 2˚ structure formation leads to local hydrophobic burial
and precedes most 3˚ interactions
 Consolidation of structure is gradual and earliest for 2˚
structure elements
Concluding Remarks. When generalized to small globular
proteins, folding proceeds by nucleation, i.e., local hydrophobic collapse of core elements, followed by consolidation of
hydrophilic interactions & 3º structural domains
© 2006, Alpay Taralp, Sabanci University
S S
S
CH3
HN
-
SH
O
O C
Native protein at slightly
alkaline pH Values
N
NH2
+
NH3
NH
C NH2
+
H2 N
NH
OH
A Closer Look at Chemical Modification
of Protein Monomers in Aqueous (and
now also Organic or Dry-state)
Environments and the Use of Papersupported Chromatography
© 2006, Alpay Taralp, Sabanci University
FOCUS
 To contrast chemical modification against enzyme
kinetics & SDM
 Discuss the applications of chemical modification
 Discuss non-destructive & destructive chemical
methods to learn about structure & function
 Look at classic derivatizations
 Discuss the advantages of competitive labeling
© 2006, Alpay Taralp, Sabanci University
Structure perturbs function,
History of chemical
function defines structure
modification (not SDM)
• Approximately the same
SH
time as enzyme kinetics
• Glutaraldehyde tanning of
Gee, my thiol must be ionized in order
leathers
to attack the peptide bond
• Refinement of foods, e.g.,
milk proteins
• Protein structure, function
and S-F studies
+
H
• Insulin primary structure
S +
His
determination, partial and
full acid hydrolysis, dansyl
There you go Mr. Thiolate! I have placed
method, cyanogen bromide
you near a positive His residue and the +
base of a large alpha-helix dipole moment!
sequence alignments,
You may attack at will!
Edman Degradation,
Carboxypeptide-MS
methods
Chemical modification and enzyme kinetics are
complementary methods.
 Advantage of enzyme kinetics:
 Enzyme bears an intrinsic probe, which allows you to examine
structure and function
 pH activity profile shows the pKa of groups that are important for
binding and/or catalysis…
 Disadvantage of enzyme kinetics:
 Not every protein is an enzyme! Other proteins which can be
bioassayed fairly conveniently are antibodies and hormonereceptor interactions.
 Advantage of chemical modification in comparison to
enzyme kinetics?
 The method can be used on any protein to determine information
related to structure and function!
 Disadvantage of chemical modification in comparison to
enzyme kinetics:
 You must insert the probe of structure and function, and you
must do it without defeating the purpose of your experiment
© 2006, Alpay Taralp, Sabanci University
Chemical modification and site-directed mutagenesis
are complementary methods.
 Rationale: Information obtained from chemical
modification forms a base to design appropriate mutants.
 E.g. Chemical modification shows that 1 of 4 Met residues is
super reactive. To learn more abot the environment, use SDM to
replace neigboring groups & observe the results…
 Advantage of chemical modification in comparison to
SDM?
 Fast, inexpensive, doesn’t require elaborate setup.
 Disadvantage of chemical modification in comparison to
SDM?
 Not very specific, and imposes a chemically reactive
environment.
 Can lead to drastic modifications.
 Modifications typically are limited to amino groups, carboxyl
groups, activated aromatic groups, sulfhydryl groups, guanidino
groups, & imidazole groups, i.e., N & C termini, glu, asp, tyr, his,
cys, met, lys, arg, trp.
© 2006, Alpay Taralp, Sabanci University
Paper methods applied to protein investigation
Historical applications – chromatography and electrophoresis
 Amino acid analysis
 Peptide mapping
 Disulfide bridge analysis
 Analysis of other post-synthetic modifications, e.g.,
phosphorylation
 Work-up of chemical modification experiments!
 Protein/peptide/amino acid resolution and purification
Current applications
 Same! Apparatus has been revised somewhat – e.g., HV tlc
is a strong alternative to HV paper methods
Advantages of paper methods over HPLC and MS methods to
identify and purify
1. Many samples can be run simultaneously
2. Multidimensional runs are conveniently setup;
sample processing is possible in between runs!
3. Resolving power is great
4. Tolerance of potential interferents is high
5. Detection methods are potentially very sensitive
and selective
6. Cost efficiency of equipment and experiments
7. Less need of skilled labor
8. MS and HPLC methods can be coupled if desired
The apparatus of paper chromatography
trough with
solvent
suspended
papers
for simple analytes
for biological samples
You may discard the drying
accessories; instead clip the
paper horizontally at the
bottom with zig-zag scissors
- allows solvent to drop
evenly to the bottom
The apparatus of high-voltage paper electrophoresis
1. paper; 2. dielectric; 3. glass tank;
4. trough with buffer (top) and base
with same buffer (bottom);
5. cathode (-); and 6. anode (+)
Types of volatile
solvent systems
Types of papers
•BAWP
•Whatmann 3MM for high
loading, large peptides
•Ammonia –
organic
•Whatmann 1MM for high
resolution
•Pyridine acetate
buffer
•Formic acid –
acetic acid buffer
Tlc alternatives as
stationary phase
•Cellulose tlc plates
•Silica tlc plates
Protein sample preparation prior to spotting on paper
Amino acid analysis
Hydrolysis (acid typically; base hydrolysis if Trp is needed)
Dry sample
Reconstitute in a minimum amount of running buffer
Spot the sample on paper
Identifying the number of arginine residues in BSA
Chemically block all lysine residues in 8M urea
Dialyze away the urea
Digest all arginyl peptide bonds using trypsin
Dry sample
Spot the sample on paper
Separating the  and b chains of insulin
Incubate protein in 95:5 formic
40V/cm
acid/30%hydrogen peroxide
Dry sample
Spot the sample on paper
e.g. for HVPE
(-)
Submerged
in buffered
trough, pH 2.1
Direction of migration
X X X X X X XXX X
Origin
Submerged
in buffer at base,
pH 2.1
Identifying and quantifying bands after
migration and drying of paper chromatograms
after migration
o
o
oo o
oo o
o
Strategies to identify
o
o
o
o o
o
o
How shall we
see these?
•Intrinsic fluorescence
•Radiolabeling and exposure of Xray film
•Fluorescent derivatization or
colorimetric derivatization, e.g.,
ninhydrin
Strategies to quantify
•Densometry of chromatographic
images
•Liberation from paper and
subsequent analysis
n-Dimensional runs & the advantage
of multi-dimensions
2-D
o
o
o
o
oo
run 1st
dimension
1-D
X X X X
X
X X
rotate
90
degrees
o
You may chemically
process the sample in
between dimensions!
run 2nd
dimension
after 1st
dimension
o
o o o oo
o
o o
o
o
o
o
o
o
o
o
2nd & subsequent dimensions
may/may not be performed
using the initial conditions
now you are interested
in this vertical series
multi-D
o
o
o
o
o
o
•Spray or Dip
you are interested
in these series
samples
o
oo
o
o o o
o o o oo
cut out,
stitch onto
new
paper!
run 2nd
dimension
o
o
o
o o
o o o
o
o
o
o
o
cut out,
stitch onto
new paper,
run another repeat as
dimension necessary
Reaction between protein functional groups & reagents
 Many protein groups interact with reagents as a function
of their ionization state
 Groups can be modified most specifically in an optimum
pH range
 At very high pH values, reaction with hydroxide ion
typically competes
© 2006, Alpay Taralp, Sabanci University
Typical reactivity
of protic moieties
as a function of pH
 Met & neutral Cys feature substantial nucleophilicity
 Carboxylic acids react with diazo compounds principally
while in the protonated form.
 Ring carbon positions on Trp, His & Tyr can be modified,
usually via electrophilic attack. Phe is not sufficiently e--rich
to promote electrophilic attack by typical protein reagents.
© 2006, Alpay Taralp, Sabanci University
Q: Are there other factors, which affect apparent
(macroscopic) reactivities? A: YES!
 Accessibility and steric considerations refer to the size &
amount of reagent used, whereabouts of reactive group,
permeation time of reaction, and the conditions of
reaction.
 Nucleophilicity considers the base strength, solvation
shell, and lone electron density, polarisability &
conjugation of centers on protein groups.
 Electrophilicity considers the electron density of protein
groups and electron deficiency of reagents.
 The “hard likes hard, soft likes soft” empirical relation
does apply to some degree. Basically, the interaction of
reacting orbitals and centers is in part determined by
their “harness” or “softness.”
© 2006, Alpay Taralp, Sabanci University
Some typical reagents of protein modification
© 2006, Alpay Taralp, Sabanci University
Some typical reagents of protein modification (contin.)
© 2006, Alpay Taralp, Sabanci University
 Structure-function studies generally follow this procedure:
 Choose to address a particular issue;
 Envisage a strategy to perturb the protein in order to investigate the
issue;
 Anticipate the best groups to modify in order to create the desired
perturbation;
 Choose the best reagent and conditions to specifically modify the
functional groups in question;
 Compare the change of properties of the modified protein to that of
the control protein;
 Form conclusions using the Okum’s Razor argument.
 Local changes and Global changes: Chemical
modifications of protein groups affect pI & pKa values,
solubility, surface hydrophilicity & hydrophobicity,
bioactivity, folding stability & global organization.
© 2006, Alpay Taralp, Sabanci University
Q: What are the principle applications of protein chemical modification?
 A: One application is to carry out structure-function studies
 Methods can be benign or destructive
 A typical protocol:
 Label the native protein
 Determine a change of property, and
 Extract useful information on the basis of the results.
 Data collected may be related to structure:
 E.g., a tyrosine specific reagent cannot react with a tyrosine residue; the
tyrosine may be buried.
 Data collected may be analytical.
 E.g., Protein X does not react with a tyrosine-specific reagent in 10M urea;
the primary sequence does not contain tyrosine.
 Data collected may be related to function and/or structure and function.
 E.g. 1., 10 Tyr residues are solvent-accessible, but 1 predominantly reacts
with trace reagent; this Tyr is unusually reactive in the protein environment.
 E.g. 2., After Lys-93 is modified chemically, substrate X shows a reduced
Km value; Lys-93 plays a role in binding. If the role is direct, this Lys is likely
near the binding site of the active center.
© 2006, Alpay Taralp, Sabanci University
NOTE - Chemical treatment of biologicals need not necessarily be destructive
and damaging. When conducting a structure-function study, your choice of
reagent and protocol should not defeat the purpose of your study
Appropriate
 Modification/
Conditions
Inappropriate
Modification/ 
Conditions
S-F problem?
Other investigative & analytical uses of protein chemical modifiers:
 Changing the net charge of the protein
 WSC and ethylenediamine
 Iodoacetic acid, succinic anhydride
 Cyclohexanedione
 Retaining the net charge but modifying the pKa
 Reductive formylation, amidination, guanidination
 Altering groups and testing their importance
 Destabilizing a protein towards denaturants or reversibly protecting a protein
 Citraconic anhydride, maleic anhydride
 Modifying protein hydrophobicity, hydrophilicity & surface activity
 Adducts with different compounds such as PEG2000
 Quantifying amino acids, functional groups, disulfide bridges, phosphate and other post-translational
modifications
 Various chemical reagents and chromatographic methods
 Chemical modification is used to re-engineer proteins for improved performance in industry.




The protein is characterized as much as possible
The improved trait is defined
A modification is envisaged to promote the property
The protein is derivatized accordingly.
© 2006, Alpay Taralp, Sabanci University
Judging if the purpose of an investigation has been
defeated
• The meaning of defeating your experimental
purpose
• Cases that are indifferent to over-reaction of
protein groups
• Cases that require post-reaction validation
Distinguishing between apparent/macroscopic
values and true/microscopic/theoretical values
• Kinetic measurements versus thermodynamic
measurements
• Measuring a value via independent methods may
give a true value
A time to chemically label and a time to
chemically work-up: Distinguishing between
the two steps
 In a protein labeling experiment and its workup, we
typically note:
Example
modifications
S S
S
HN
CH3
SH
O
O
O C
N CH3
H
N
NH
C NH2
H2+N
NH
O
H3C
O
 Acetylation using Acetic Anhydride or Acetyl Imidazole at pH 7.5-9
 Acetylation blocks free amino termini, lysine side-chains and tyrosine sidechains. Acetyl tyrosine is hydrolyzed at high pH values or transesterified
above pH 6 by a strong nucleophile.
 Acetylation of amino groups is usually quantitative only if the reaction is
carried out in urea solution. Without urea, usually 60% of the lysine residues
are modified.
 This modification could have the following effects
 Decrease of protein solubility (Acetyl BSA is only soluble at pH < 5);
 Changes in biological activity (by either global structural changes or specific
changes in the active site);
 Dissociation of multimeric complexes (if the surface charges are required for
association to other biomolecules).
© 2006, Alpay Taralp, Sabanci University
 Succinylation using Succinic Anhydride at pH > 7
 Succinylation blocks amino groups without modifying other functional
groups.
 Succinyl tyrosine is hydrolyzed above pH 5 via an intramolecular
cyclization.
 The effect of succinylation parallels some of those described above for
acetylation.
 Succinylation is typically used to improve the solubility of poorly
soluble proteins, particularly at pH values above 5.
 Succinylation induces separation of protein aggregates (eg.
Hemerythrin dissociates into eight subunits).
 Succinyl proteins usually unfold more easily and demonstrate a shift in
their pH optima (assuming that activity is retained).
 Succinylation is often used as a linker molecule through which the
protein be attached to a foreign surface.
© 2006, Alpay Taralp, Sabanci University
H
S S
S
HN
CH3
S
O
O C
sometimes a
little bit here
O
OO-
O
O
N CH CHCOOH
S S
HN
NH
C NH2
H2+N
NH
CH3
SH
O
O
O C
CH3
N CH CCOOH
N
N
S
NH
C NH2
H2+N
NH
OH
OH
 Maleiylation (left) using maleic anhydride & citraconylation
(right) using citraconic anhydride at pH > 8
 Maleic anhydride and citraconic anhydride block amino groups but
can be removed by incubating the protein at low pH.
 Maleic anhydride is more difficult to remove & sometimes leads to
a minor side-reaction, in which Cys adds across the double bond.
 Citraconic anhydride is used to reversibly block amino groups and
to protect the amino groups from other chemical reactions.
 E.g., citraconylation will protect amino groups from subsequent oxidation by
hydrogen peroxide. Once Cys’s are oxidized, amino groups are regenerated.
 Citraconylation is the method of choice to temporarily solubilize
poorly soluble proteins.
© 2006, Alpay Taralp, Sabanci University
 Polypeptidylation using carboxyanhydrides at pH 7
 Carboxyanhydrides add onto amino groups, releasing carbon
dioxide and producing new amino groups.
 In presence of excess reagent, the reaction repeats, building a long
polypeptide chain that extends into solution.
 Technique can improve solubility of insoluble proteins (R = H) &
conversely, reduce the solubility of soluble proteins (R = i-propyl).
 Polyvalylribonuclease, for example, aggregates in solution above 30C
 Used to study hydrophobic interactions between proteins.
© 2006, Alpay Taralp, Sabanci University
S S
HN
S
CH3
SH
O
O
O C
N CF3
H
N
NH
C NH2
+
H2 N
NH
OH
 Trifluoroacetylation using Ethyl Thiotrifluoroacetate at pH 10
 Ethyl thiotrifluoroacetate is used to reversibly block amino groups as in the case of
maleic anhydride and citraconic anhydride.
 But the derivative has a net zero charge. Furthermore, the group is removed under basic
conditions using sodium carbonate at pH 10.7 or 1M piperidine.
 E.g. 1., pancreatic ribonuclease was deactivated completely following trifluoroacetylation
yet there were no measurable structural changes. In carbonate buffer, activity was
gradually restored.
 E.g. 2., trifluoroacetylation caused structural changes in cytochrome c. Incubation in
carbonate buffer restored full electron transfer ability.
 Trifluoroacetylation can simplify protein sequencing if trypsin is used to cleave the
protein
 Trypsin normally cleaves after every free arginine and lysine residue, generating a complicated
mixture. If the protein analyte is first trifluoroacetylated, trypsin can only cleave at the arginine
bonds.
 The fragments are isolated, deblocked and subjected to a 2nd tryptic digestion at the lysine sites
© 2006, Alpay Taralp, Sabanci University
S S
HN
S
CH3
S S
SH
HN
S
CH3
SH
+
N H2
O
O C
N CH3
H
N
NH
C NH2
H2+N
NH
-
N+H2
O
O C
N NH2
H
N
NH
C NH2
H2+N
NH
OH
OH
 Amidination using methylacetimidate (left) or guanidination
using o-methylisourea (right) at pH 7-10.
 Methylacetimidate forms stable derivatives with lysine residues
and amino termini. O-methylisourea reacts with lysine groups.
 Modification of amino groups increases steric bulk, while retaining the
positive charge. The pKa of derivatives shifts to values well above 11!
 Most modifications do not give significant structural changes.
 Q: Suggest a test to postulate if the active-center lysine is the
catalytic nucleophile or a binding group.
 Q: Suggest another test to determine if a lysine and a neighboring
aspartic acid maintain a crucial salt bridge.
 Hint: You will analyze the pH activity after reaction.
© 2006, Alpay Taralp, Sabanci University
S S
HN
S
CH3
SH
O
O
O C
O
N OCH2CH3
H
H3CH2CO N
NH
C NH2
+
H2 N
N
OH
 Ethoxyformylation using diethylpyrocarbonate
(ethoxyformic anhydride) at pH 4
 Ethoxyformic anhydride only reacts with imidazole groups at pH 4; it modifies
amino groups at basic pH values.
 The acyl-imidazole adduct is stable in water, particularly at pH 7, unlike other
acyl His derivatives. The group is removed by the action of H2N-OH at pH 7.
 EFA is used in molecular biology to battle against RNAse.
 EFA can rapidly inactivate many enzymes including trypsin and is used by
industry to cold-sterilize food.
 EFA can aid structure-function studies if histidine is important for bioactivity.
 Q: You must decide if a Lys or a His is essential for catalysis. What experiment could
you design using the reagents we have discussed thusfar?
© 2006, Alpay Taralp, Sabanci University
S S
HN
S
S S
CH3
HN
SH
O
O C
CH3
+
NH
CH3
N
NH
C NH2
H2+N
NH
S+(CH3)2
S+(CH3)2
O
O C
CH3
N CH3
CH3
-
+
H3C
N+
NH
C NH2
+
H2 N
N
CH3
OCH3
OH
 Reductive dimethylation using H2C=O & NaBH4 (left) at
pH 9; Variable methylation using ICH3 (right) at pH 2-10
 Reductive methylation mono/dimethylates amino groups, retaining the positive
charge of the amine.
 Generally, structural changes are not observed following reductive methylation.
 Reaction with iodomethane quaternizes amino groups with retention of positive
charge. Met and Cys are converted to their sulfonium iodides. Tyr is methylated
and His is converted to the dimethylimidazolium iodide.
 Reaction selectivity is tuned by appropriate choice of pH & reaction medium.
 Unlike reductive methylation, iodomethane:
 Puts a permanent positive charge on amino, imidazole and sulfide groups
 Removes the hydrogen bonding ability of tyrosine
© 2006, Alpay Taralp, Sabanci University
H3C
S S
S+CH2COOS+(CH2COO-)2
HN
CH2COO+
NH
CH2COO-
O
O C
-
OOCH2C
N+
N
CH2
COO-
NH
C NH2
H2+N
H3C
S S
S+CH2CONH2
S+(CH2CONH2)2
HN
O
O C
CH2CONH2
NH
CH2CONH2
-
+
H2NOCH2C
N+
OH
N
CH2
CONH2
NH
C NH2
H2+N
OH
 Carboxyalkylation using iodoacetate (left) & carbaminoalkylation using iodoacetamide (right) at pH 2-10.
 Iodoacetate & iodoacetamide react similarly to iodomethane
except tyrosines are generally not modified.
 Derivatives are larger than iodomethane
 Derivatives may be considered bulkier.
 In the case of iodoacetic acid, a negative moiety is introduced.
 Iodoacetamide, iodoacetic acid and iodomethane are commercially
available in NMR-active and radioactive forms.
© 2006, Alpay Taralp, Sabanci University
S
S
HN
S
S
CH3
S
HN
NH2
N
O
H3CO C
NH
C NH2
+
H2 N
NH
CH3
SH
SH
O
O
H2NCCH2O C
S
NH2
N
NH
C NH2
+
H2 N
NH
OH
OH
Esterification using diazoglycinamide at pH 5 (left)
or 0.1M methanolic HCl (right).
Reactions are acid catalyzed
With diazoglycinamide, the strongest proton donors (Cterminal carboxylic acids) react first
With methanolic HCl, esterification hastens as [HCl] increases.
 Esterifıcation time course experiments often correlate to a loss of
biological activity
© 2006, Alpay Taralp, Sabanci University
S
S
HN
S
CH3
SH
O
HOCH2CH2NH C
NH2
N
NH
C NH2
H2+N
NH
OH
Amidation using amine, hydroxysuccinimide, and
ethanolamine at pH 4.75
Amidation is a general method of converting protein into
many useful forms.
By appropriate choice of amine, a positive, zero or negative
charge can be introduced at the carboxylic acid sites.
E.g. 1., immobilization of enzyme has been carried out using
carboxyl functions.
E.g. 2., chemical modification can be used to probe the active
site carboxylates of pepsin.
© 2006, Alpay Taralp, Sabanci University
SH SH S CH3
HN
SH
O
HO C
NH2
N
NH
C NH2
+
H2 N
NH
OH
 Reduction using mercaptoethanol or dithiothreitol (reduced
form) at pH 8.
 Cystine bridges can be cleaved to afford two cysteines by the
action of mercaptoethanol or dithiothreitol.
 In the case of DTT, a stoichiometric amount of reagent is sufficient to bring
about the modification. The driving force for the reaction is formation of a
stable 6-membered ring.
 Reduction has been used to study bioactivity & structure.
 In many cases, reduced proteins exhibit changes of solubility & activity.
 Reoxidation of reduced cysteines sometimes reforms the correct disulfide
bridges & restores structure and function.
© 2006, Alpay Taralp, Sabanci University
Apparent relative reactivity and pK a values of 3 protein
groups with respect to an external Phe-NH 2 standard
Competitive Labeling
Many methods used to
modify different functional
groups
1-
Generally the modifier is
used in excess → proteins
modified extensively.
Onus is put on the
investigator to prove that the
results reflect the properties
of the native protein.
Molar
radiolabel
incorporation
(apparent
0.5relative
reactivity)
with respect
to Phe-NH2
apparent pK a
of reactive group
pH value of reaction
 In competitive labeling, a modification ALWAYS reflects the state of the
native protein. Why?
 Reagent is used in trace amounts
 At most 1 group/protein is modified
 Thus, only the reactivity of the native protein is probed
 Above: A typical experiment quantifies the statistical distribution of the kinetic
reactivity of protein groups
 the reactivity of every reactable group is probed at different pH values.
 While the results describe apparent data, they are used to interpolate a group’s
local environment.
© 2006, Alpay Taralp, Sabanci University
 Steps: 1.Tracer. Incubate a solution containing protein & standard (e.g.,
Phe-NH2) with a trace amount of 3H-reagent. Tritium incorporation is your
probe. Carry out this operation at many pH values.
 2.Normalization. Unfold the protein in urea and react the remaining groups
completely with 14C labeled reagent. This protocol ensures that all protein
and standard derivatives become chemically homogeneous.
 3.Quantification. Extract the standard quantitatively into an organic phase
and quantify the 3H/14C ratio for each pH value. Digest the protein using
enzymes so that only one modified group is present per peptide. Migrate all
peptides in an electric field. Identify peptide positions using
autoradiography. Collect each band & quantify the 3H/14C ratio. Sequence
the peptide in order to identify the derivative.
 4. Interpretation. The 3H/14C ratio of protein & standard are compared. The
reactivity of the standard reflects its aqueous environment & should parallel
its titration curve. The reaction profile reflects the groups’ local environment
so you should observe a “titration curve”, which illustrates the effect of the
protein environment. This titration curve may deviate significantly from
what would be expected in an aqueous environment. The data can thus be
used to build a picture of the local environment.
© 2006, Alpay Taralp, Sabanci University
 Two data can be extracted for every group: The kinetic pKa of the group &
the relative reactivity of the group can be assessed in comparison to PheNH2, which has a well-characterized reactivity and pKa that is devoid of any
environmental influences. The two data help to assess if any potential steric
considerations or stereoelectronic factors are out of the ordinary.
 Below are some example results, which allow you to appreciate the power of
this method:
 e.g. Titration curve of Cys gives a pKa of 3. Perturbed pKa.
 e.g. Titration of Lys-29 gives poor reaction when reacted from pH 5 to
11. Buried.
 e.g. Titration of Lys-29 gives a continuous titration curve with an
interpolated pKa of 11 when reacted from pH 5 to 11 (A normal lysine is
10.5). Lysine is buried part of the time or otherwise perturbed.
 e.g. Titration of Lys-29 gives a discontinuous curve when reacted from
pH 5 to 11. First there is no reaction, then after pH 10.5 the reaction is
very high. Conformational change and accessibility.
 e.g. The N-terminal Histidine imidazole ring displays a pKa value that is
equal to the pKa of its N-terminal amino group. Inductive effects and
coupled reactivities.
 e.g. Topography of E.coli Ribosomal Protein L12 in situ Eur. J.
Biochem 80, 35-41 (1977). Next slide (please).
© 2006, Alpay Taralp, Sabanci University
The power of competitive labeling and paper methods
© 2006, Alpay Taralp, Sabanci University
RECAP - Steps of competitive labeling: 1. Label each
protein at most once, using a trace of tritiated reagent
-3
Me H*
S
H3C
S
His
H3C
95 *3-Me-OH
1000
His
NH2
997
+100 *3-Me-I
in a pH "X" solution
(aq)
Tyr
Standard, with known pK a value & reactivity!
S
S
S
H3C
H3C
His
1
NH2
-3
Me H*
+
1
Lys
NH
+
-3
Me H*
(aq)
Tyr
999 AcHisNH2
Lys
+
+
H3C
His
(aq)
Tyr
Lys
Tyr
1000 AcHisNH2
+
(aq)
Lys
+
NH2
+
-3
Me H*
His
1
NH2
(aq)
Tyr
1 AcHisNH2
-3
Me H*
Lys
Step 2: Label all protein completely using excess low
specific actitivity 14C-MeI to yield a chemically homogeneous mixture
99% * MeOH/ MeOH
14-
12-
+excess *14-Me-I/ 12-MeI mixture
in 8M urea pH 10 solution
-3
Me H*
S
S
H3C
H3C
His
997
NH2
1
His
NH2
+
997
Me
Me
(aq)
+
Me
S
+
S
S
H3C
His
+
1
Me
His
Lys
1
NH2
+
1
Me
Me
S
Tyr
Me
+
999 AcHisNH2
-3H*
N
Me
Me
(aq)
Lys
1
NH
+
Tyr
Lys
Me
+
1 AcHisNH2
-3
Me H*
-3
Me H*
+
+
Tyr
Me
+
999 Ac HisNH2
Me Me
+
-3
Me H* Me
Me
His
1
+
Me
Me
N
Me (aq)
(aq)
Lys
Me
Me
+
Me
+
Me
Me
Me
(aq)
+
H3C
-3
Me H*
N
Me
Me
+
H3C
+
Tyr
Me
Me
Me
His
Me
Lys
Tyr
+
H3C
Me
N
(aq)
Lys
Tyr
His
S
Me
+
H3C
Me Me-3H*
+
Me
(aq)
H3C
Me
Me
S
His
+
Lys
Tyr
Me
+
Me
Tyr
+
Lys
+
Me
1 Ac+HisNH2
-3
Me Me H*
(aq)
Me
Subsequent Steps (for proteins of known 1˚ sequence):
Step 3. Repeat steps 1 & 2 using more native protein; label many
samples at different pH values.
Step 4. Prepare a positional marker protein; label native protein
with high-specific activity 14C-MeI, then excess 12C-MeI in 8M urea
Step 4. Digest all methyl-proteins separately. Use 2-4 proteases.
Step 5. Separate the different peptides in an electric field along tlc
or paper; use 1-4 dimensions; spot the marker peptides at the
ends of the chromatogram.
Step 6. Expose X-ray film to the chromatogram (RT for 14C, -80˚C
for 3H) to find the position of all substantially radioactive peptides.
Step 7. Find the 3H/14C ratio of each peptide band; sequence each
to identify the reactive group (e.g., AlaTyr*Gln can only be Tyr18)
Step 8. Plot the reactivity (i.e., 3H/14C ratio) of each reactive group
as a function of pH.
Paper chromatogram beneath;
cathode (-) X-ray film superimposed on top
.
9
.
Recover & measure
3 H cpm/ 14C cpm
8
7
e.g.1; peptide 8 @ pH 7.8 = 512/1890
6
e.g.2; peptide 7 @ pH 7.6 = 450/5680
e.g.3; peptide 6 @ pH 8.1 = 890/1543
3kV,
30min, 5
pH 2.1
4
.
3
2
1
x x x x x x x x x x x x x x xx x
.
.
7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.18.2 8.3 8.48.5
pH of labeling
anode (+)
14C-marker peptides
.
Peptide 6 was sequenced:
AlaTyr*Gln indicates a Tyr
reacted. There are 4 tyrosyl
residues in this protein but the
sequence data indicates Tyr18
= radioactive ink spotted on
Whatmann 3MM paper
Peptide solutions spotted
along paper at origin
Apparent relative reactivity and pK a values of 5 protein
groups with respect to an external AcHis-NH 2 standard
His102
1Molar 3H/ 14 C
radiolabel
incorporation
(apparent
0.5relative
reactivity)
with respect
to AcHis-NH 2
AcHis-NH 2
Lys 87
Tyr 18
NH 2
Met1
apparent pK a
of reactive group
pH value of reaction
(referenced against known
pKa of AcHis-NH 2)
In vitro manipulatin of protein
monomers or their environments to
enhance performance
© 2006, Alpay Taralp, Sabanci University
Goals: To formulate broad-scope protein preparations, which are:
 Cheaper
 More stable
 More catalytic
 Longer-lived
 More easily stored
& transported
 More active at pH &
temperature extremes
Approach 1: use proteins
in water-free media
Approach 2: crosslink
proteins together
search for robustness:
Locating/purifying thermophiles, etc.
Native
Low-tech
chemical strategies
Approach 3: chemically
modify protein monomers
© 2006, Alpay Taralp, Sabanci University
Genetic
manipulation
As scientists, you will occasionally prepare proteins, which are not in water.
Please note the following pharmaceutical example:
Insulin
intraperitoneal injections
time-released matrix
İnhalable microcapsules
Lysozyme
creams & gels
inhalable microcapsules
drop formulations
Question: What are some biases related to proteins in unusual environments?
Fact 1: Protein structure & function is sensitive to pH, temperature, ionic
strength, etc.
Fact 2: Eminent scientists have said “Enzymes need aqueous environments to
function because this is how Nature has intended them to function.”
Question: In your profession, you may prepare proteins in unusual
environments. Should you worry?
© 2006, Alpay Taralp, Sabanci University
Answer: NO! Recent evidence shows that protein structure and protein function
may be very tolerant to strange environments! 
Today, we will discuss the structure & function of proteins in reduced-water &
water-free environments
Why does mankind wish to use biologically active proteins?
Proteins accelerate chemical reactions
Proteins improve product properties
Proteins permit novel syntheses
Here are some typical industrial applications:
Bioreactors
Textile treatment
Medicinal and organic syntheses
Protein drugs & drug delivery
Biosensors
Bioremediation
Food preparation industries
Problem? Industrial conditions are often too harsh for proteins in the native state.
Consequences: Poor biological activity, short lifespan, limited reaction parameters,
etc.
© 2006, Alpay Taralp, Sabanci University
The new view: Proteins can maintain their structure & function
without an aqueous environment.
Implication? Your protein drug formulations may be
perfectly happy in a gel, or a cream or in a sugar-coated
matrix
Example I: Rate comparisons in octane
Enzyme kcat/Km (M-1s-1)
Chymotrypsin
Subtilisin
Rate (kENZ)
0.7
1.8
Rate(kNONENZ)
1.1 x 10-11
1.1 x 10-11
Enhancement
6.4 x 1010
1.6 x 1010
N-Ac-L-Phe-OEt + amyl alcohol → N-Ac-L-Phe-OAmyl + EtOH in octane
NOTE – Reactions in water are much faster!
Typically kcat/Km water / kcat/Km octane = 104-107
Q: Can we improve the speed of an enzyme reaction in octane?
A: YES! But first we must understand more...
© 2006, Alpay Taralp, Sabanci University
Example II: Protein Structure & Integrity
o
o
H2O
H2O
o
H2O
o
H2O
o
o
H2O
o
H2O
organics
o
H2O
H2O
o
organics
organics
H2O
H2O
H2O
H2O
o
o
H2O
H2O
organics
organics
organics
organics
organics
o
o
organics
o
o
organics
organics
organics
o
organics
o
organics
organics
organics
o
Q: -Momorcharin was crystallized in
water (thin lines), then crosslinked, &
solvent-exchanged with CH3CN (thick
lines). Compare the structures.
Similar results were obtained with:
 chymotrypsin in hexane
 subtilisn carlsberg in acetonitrile
© 2006, Alpay Taralp, Sabanci University
Example III: Solvent Effect on Catalytic Rates
Lyophilized (pH 7.8) chymotrypsin + dry organic solvent  CT suspension
Lyophilized (pH 7.8) subtilisin + dry organic solvent  SBL suspension
N-Ac-L-Phe-OEt + n-propanol  N-Ac-L-Phe-n-OPr + EtOH
Solvent
Hexadecane
Octane
Carbon tetrachloride
Toluene
Ethyl Ether
Acetone
Acetonitrile
Dimethylformamide
Dimethylsulfoxide
Vmax/Km (min-1 x 10-6)
Subtilisin Chymotrypsin
3900
4300
2000
1700
340
96
150
120
97
48
810
0.6
150
0.4
19
<0.1
<0.1
<0.1
Q: Is the above data incorrect?
A: NO! Products were NEVER formed unless the enzyme was added!
© 2006, Alpay Taralp, Sabanci University
Question: How do we explain this trend?
Answer: Hydrophilic organic solvents strip essential water from the enzyme.
What is the evidence?
 Enzyme activity correlates directly with the amount of water retained by
the “dry” enzyme
 Enzyme activity improves greatly by adding small amounts of water (1.5%)
to the hydrophilic organic solvents.
Example IV: Active Site Integrity
Q: Chymotrypsin(dispersed in octane) + phenylmethylsulfonyl fluoride
yielded inactive enzyme. What is implied?
SO2 O
Ser in active site
© 2006, Alpay Taralp, Sabanci University
Example V: Rate Measurements in octane
A
Start
E+S
k1
k-1
E
ES
B
P
F
k2
E+P
Q
E
[ E]o
v
Q: Rate measurements in
organic solvents behaved
as if in aqueous solution!
What is implied?
© 2006, Alpay Taralp, Sabanci University
Finish
= Fo +
1/v
FA
FB
+
[ A]o
[ B ]o
B3
B2
B1
1/[A]o
organics
o
o
organics
organics
organics
o
o
organics
o
organics
organics
o
organics
o
o
organics
organics
o
organics
Example VI: pH
activity
Q: Chymotrypsin activity in
octane depends on the pH
of solution from which
the enzyme was dried.
Why?
A: pH “memory”
Choice 1: set pH here and
lyophilize
kcat/Km
pH
Lyophilize
Choice 2: lyophilize and then
optimize pH with organic
soluble buffers
kcat/Km
© 2006, Alpay Taralp, Sabanci University
LpH
Example VII: Imprinting protein in the dry state
Q: Enzyme dried in the
presence of acetyl-Lphenylalanine was more
active. Why?
A: Positional imprinting of
active site groups!
o
o
o
o
o
o
o
o
o
© 2006, Alpay Taralp, Sabanci University
Example VIII: Competitive inhibition of
chymotrypsin in water & in octane
Inhibitor
Inhibition Constant KI(mM)
In water
In octane
Benzene
21
1000
Benzoic Acid
140
40
Toluene
12
1200
Phenylacetic acid 160
25
Naphthalene
0.4
1100
1-Naphthoic acid 7.2
3
Q: Good competitive inhibitors in water are poor competitive
inhibitors in octane! Poor competitive inhibitors in water are
excellent competitive inhibitors in octane! WHY?
© 2006, Alpay Taralp, Sabanci University
Example IX: Substrate specificity (kcat/Km) of
chymotrypsin & subtilisin (in water or octane)
Chymotrypsin
Subtilisin
Substrate
hydrolysis transesterification
hydrolysis transesterification
N-Ac-L-Phe-OEt
4.00 x 104
0.72
1.3 x 104
1.7
N-Ac-L-His-OMe
2.00 x 102
1.5
5.5 x 102
3.1
N-Ac-L-Ser-OMe0.87 x 102
2.5
1.6 x 102
4.5
Q: Hydrophobic groups yield better substrates in
water! Hydrophilic groups yield better
substrates in octane! WHY?
© 2006, Alpay Taralp, Sabanci University
Example X: Rates and enantioselectivities of
propanol/N-Ac-Phe-OEt transesterification using
Aspergillus oryzae protease in anhydrous solvent
Initial Rate (mmol h-1)/mg protein
Solvent
L-enantiomer
D-enantiomer
enantioselectivity
(vL/vD)
acetonitrile
0.85
0.12
7.1
pyridine
0.645
0.15
4.3
acetone
0.54
0.41
1.3
dichloromethane
0.29
0.33
0.88
methyl tert-butyl
2.2
6.4
0.34
octane
2.9
12
0.24
tetrachloromethane 1.7
8.9
0.19
Q: Acetyl-L-Phe-OEt is a better substrate in polar organic
solvents, whereas acetyl-D-Phe-OEt is a better substrate in
very hydrophobic solvents. WHY?
© 2006, Alpay Taralp, Sabanci University
Q: Protein structure & function may suffer in proceeding
from water to dry environments. How can we help?
o
o
o
o
o
o
o
o
o
© 2006, Alpay Taralp, Sabanci University
A linear profile
(black) indicates
that diffusion is
not rate-limiting.
A convex profile
(red) would be
expected if
diffusion was an
important factor.
the fraction (f) of the active subtilisin in the CLCs
o
o
o
o
o
o
o
o
o
© 2006, Alpay Taralp, Sabanci University
In summary, when proteins are put into unusual
environments they can survive!
Final Question: What are some advantages of
using enzymes in water-free environments?
 Novel reactions, which are not feasible in water, become posssible
Use of “non-water” nucleophiles, e.g. transesterification and
ester ammonolysis;
Increased solubility of apolar substrates; and
shifting thermodynamic equilibria to favor synthesis over
hydrolysis, e.g., esterification and peptide formation.
 Suppression of water-mediated side-reactions.
 Alteration of substrate specificity.
 Enhanced thermostability of enzymes.
 Easy recovery of enzyme from low boiling solvents.
© 2006, Alpay Taralp, Sabanci University
Crosslinking Protein Solids to
Enhance Utility
© 2006, Alpay Taralp, Sabanci University
You may prepare protein drug formulations, which are not in
water:
We saw some examples just before:
Insulin
Lysozyme
intraperitoneal injections
creams & gels
time-released matrix
inhalable microcapsules
İnhalable microcapsules
drop formulations
New Facts: Protein structure & function can be stable in unusual
environments if you use the correct procedure! What Are The
Results????
 Cheaper
 More stable
 More catalytic
 Longer-lived
 More easily stored & transported
 More active at pH & temperature extremes
Question: In your profession, you might wish to prepare “super”
proteins, which are very effective in various applications. What are
your available strategies? © 2006, Alpay Taralp, Sabanci University
Strategies to engineer better proteins
Locating/purifying thermophiles, etc.
Approach 1: use proteins
in water-free media
Approach 2: crosslink
proteins together
Native
Low-tech
chemical strategies
Approach 3: chemically
modify protein monomers
© 2006, Alpay Taralp, Sabanci University
Genetic
manipulation
Approach 2: Crosslinking of protein
Crosslinking of protein solids
Q: Why crosslink protein?
Improved structural
stability
Potentially altered
function
Native
Crosslinked enzyme crystals
Solution phase crosslinked enzymes
© 2006, Alpay Taralp, Sabanci University
Established crosslinking media:
A. In a solubilizing environment
Q: What are the characteristics of method A?
Solution-phase crosslinking often leads to polydispersity
Events: Soluble monomers → Dimerization →
Oligomerization →→→ Insolubilization (typically gelation)
© 2006, Alpay Taralp, Sabanci University
B: In the crystalline state
Q: What are the characteristics of method B?
Crosslinked enzyme crystals are biologically
active catalysts with well-defined pores
Events: Soluble monomers → Crystallization
→ Chemical crosslinking →→→ Insolubilization
© 2006, Alpay Taralp, Sabanci University
C: In the lyophilized state
Q: What are the characteristics of method C?
Crosslinking of lyophilisates leads to polydispersity
Events: soluble monomers → lyophilization at desired pH value
→ vacuum, heat and/or chemical crosslinker → oligomerization
→→ insolubilization (powder resists swelling in solvents)
© 2006, Alpay Taralp, Sabanci University
D: As a precipitate retaining the native
structure
Q: What are the characteristics of method D?
Events: Soluble monomers → Salt-out or insolubilize with
miscible organics → Add chemical crosslinker → Oligomerization
→→ Insolubilization (powder resists swelling in solvents)
© 2006, Alpay Taralp, Sabanci University
E. Supported enzyme technologies
Q: What are the characteristics of method E?
Surface-immobilized enzymes
Surface-immobilized
enzyme aggregates
Crosslinked and
intertwined enzymes
© 2006, Alpay Taralp, Sabanci University
Q: How do the proteins eventually combine together?
Method A: Classic solution-phase crosslinking of dissolved proteins
© 2006, Alpay Taralp, Sabanci University
Q: Why is crosslinking solution-phase proteins (Method A) not
equivalent to crosslinking of “pre-solidified” proteins (Methods B-D)?
Method A: In the solution phase,
all enzymes have equivalent
chances of reacting at time t = 0.
When reactive chemical crosslinkers are employed, the
outer enzymes preferentially react at time t = 0 → teq.
Solution phase
Methods C & D
Method B
Amorphous solid
(enzyme aggregate
or lyophilisate) © 2006, Alpay Taralp, Sabanci University
Enzyme crystal
Q: How are proteins bonded together?
A: Chemical (& thermal) strategies
Common reagents: glutaraldehyde,
The physico-chemical properties of the
glyoxal, glycolaldehyde, formaldehyde,
interprotein bond is variable:
WS carbodiimide, bifunctionals such as
I. Length: Crosslink varies from zero length
alkyldiimidates, diisoureas, diketohalides,
to multi-carbon units
disulfonates & ditresylates, cyanuric Cl
II.
Reagent-free, thermal crosslink induction:
www.proteovak.com
[
O
]n
N
O
s
?
S
S
N
S
?
Bonding is generally mediated by the
protein amino groups and carboxyl
groups. Cystine bonds are also important.
III. Some reactions proceed with charge
retention of the bonded groups, whereas
other reactions proceed with a change.
+
O2N
H
C
NO2
NH
N
N
H
H
NH
NH
+
N H2
NH
O
NH
+
N H2 N H2
H
O
N
(CH2)n
+
+
N
N H2
O
N
O
O
+
N H2
(CH2)n
NH
N
+
N H2
+
N H2
© 2006, Alpay Taralp, Sabanci University
Q: Let us please summarize approach 2: protocols, reagents & products
A. In aqueous solution
B: In the crystalline state
D: As a precipitate retaining the
native structure
Your choice of reagent? Prior history; Surface
accessibility and steric constraints; &
effective pH during the reaction conditions
A: glutaraldehyde, other simple aldehydes,
EDC.HCl, diimidates, difluorodinitrobenzene
B: glutaraldehyde, other simple aldehydes
C: In the lyophilized state
© 2006, Alpay Taralp, Sabanci University
C: volatile or organic soluble carbodiimides,
acyl group activators, disulfide exchange,
thermal induction of amide bonding,
bifunctional acyl halides and similar reagents
D: glutaraldehyde, dextran polyaldehyde
S S
S
CH3
HN
-
SH
O
O C
Native protein at slightly
alkaline pH Values
N
NH2
+
NH3
NH
C NH2
+
H2 N
NH
OH
Re-engineering Protein Monomers via
Chemical Modification in Aqueous,
Organic or Dry Environments
© 2006, Alpay Taralp, Sabanci University
Review of Approach 3: Chemical modification of proteins to alter properties
modify the enzyme
in specific areas
new enzyzme is faster or
new enzyme is stabler or
new enzyme has different pH activity
 Q: What are some chemicals to alter protein charge?
 Ac2O, MeI, succinic anhydride, H2NCH2CH2NH2/ carbodiimide, iodoacetic
acid
 Q: What are some chemicals to alter protein hydrophobicity?
 Octadecyliodide, PEG-tresylate
 Q: What are some reaction environments to alter protein
groups?
 Aqueous reagents acting of dissolved protein
 Organic-phase reagents acting on protein powder
 Vapor phase reagents acting on protein powder
 Let us examine the fate of one enzyme, whose charges were
altered in water using chemical reagents
© 2006, Alpay Taralp, Sabanci University
COO
-
COO
-
-
-
OOC
OOC
+
-
NH3
example
-
OOC
inactive
Q: In this hypothetical
example, is the
enzyme active at low
or higher pH values?
COO
OH
-
NH2
-
OOC
active
catalytic
rate
Q: How do you
rationalize the
difference?
pH 7.5
pH
© 2006, Alpay Taralp, Sabanci University
COO
-
COO
-
COO
-
-
OOC
+
-
OOC
COO
OOC
-
NH3
OH
-
NH2
-
OOC
inactive
O
+
O
+
CED
H
+
NH3
OC
ED
COO
-
active
H2NCH2CH2NC
+
-
inactive
ethylene diamine + carbodiimide
O
+
O
CED
+
EDC
OH
NH2
CO
OC
+
ED
+
ED
active
Q: What happens to the charge distribution along the surface?
© 2006, Alpay Taralp, Sabanci University
CO
+
ED
Q: In this hypothetical example
(left), what has happened to the
pH activity of your antimicrobial
protein drug (two things)? Please
rationalize...
catalytic
rate
pKa 7.5
Q: Is this change desirable (local
pH of infected regions is lower)?
pKa 6.5
pH
Gibbs
energy
Q: You may have obtained a
desirable change, but many times
the change is not free... What
price have you paid in this
example (right graph)?
Eu Eu
EF
EF
© 2006, Alpay Taralp, Sabanci University
reaction coordinate
Q: So where do we stand?
User-friendly protocols to aggregate
& crosslink protein and to improve stability
Know-how to use proteins
in water-free environments
Chemical agents to alter
protein groups & function
© 2006, Alpay Taralp, Sabanci University
Thus...
My suggestion
 Combine the 3 strategies to enhance the performance of
proteins as drugs, etc., and as enzymes
Examples of heat-crosslinked
protein & analysis
© 2006, Alpay Taralp, Sabanci University
Addition to Notes:
Protein Purification &
Related Analytical
Methods
Some References
Harris, E.L.V. and Angal, S. eds., Protein Purification Methods: A
Practical Approach, 1989, IRL Press
Harris, E.L.V. and Angal, S. eds., Protein Purification Applications: A
Practical Approach, 1990, IRL Press
Deutsher, M.P., ed., Methods in Enzymology, Guide to Protein
Purification, 1990, Academic Press
© 2006, Alpay Taralp, Sabanci University
If you wish to obtain new proteins, you must understand how to purify &
test these proteins
Three protein types:
Membrane
Intracellular
Extracellular
© 2006, Alpay Taralp, Sabanci University
Disruption
Organelle Isolation
Intracellular Protein
Disruption/Solubilization
Clarification
Centrifugation/flocculation or Liquid 2-Phase Partitioning
Liquid 2-Phase Partitioning
Ammonium Sulphate Precipitation
Primary Separation Techniques
Organic Solvent
Precipitation
Chromatography Techniques
Ion Exchange
Affinity
Metal Chelate
Covalent
Hydrophobic Interaction
Ion Exchange
Chromatofocussing
Hydrophobic
Interaction
Other Absorbtion
Methods
Gel Permeation
© 2006, Alpay Taralp, Sabanci University
© 2006, Alpay Taralp, Sabanci University
To purify a protein, here are some general rules:
1. The following methods are used to purify protein:
Technique
pH Precipitation
Ammonium Sulphate Precipitation
Ion exchange chromatography
Hydrophobic Interaction chromatography
Chromatofocusing
Dye Affinity chromatography
Ligand Affinity chromatography
Gel permeation chromatography
Exploited Protein Property
Charge, pI value
Intermolecular charge/
hydrophobic interactions
Charge
Hydrophobicity
Charge, pI value
Affinity for high MW dye
Bioactivity/affininty
Dynamic volume (size)
2. No single method is perfect, so you should use many together 
Example manipulation:
Starting purity = 10%, yield is 10mg of target protein
After AS precipitation, purity = 60%, yield is 8mg of target protein
After Chromatography, purity = 95%, yield is 4mg of target protein
3. Protein is characterized after purification: Final yield? MW? N- & C-terminal
analysis, disulfide bridge analysis, etc.
© 2006, Alpay Taralp, Sabanci University
OH
HO
N
HN
NGlycine
S
S
S
S
NH
NValine
N
OH
Your target is insulin
pI = 4.5, MW = 5600
© 2006, Alpay Taralp, Sabanci University
OH
With recombinant Human insulin:
Lysis the cells, centrifuge, & collect your sample:
A solution of insulin, other proteins, DNA, salts, metal ions,
etc. You deterimine 1% insulin, 99% other proteins
and a total of 2g insulin in the batch
SDS gel analysis
How to purify?
Centrifuge lysed insulin
pH pptation
AS pptation
ion exchange
hydrophobic chromatography
insulin receptor column
© 2006, Alpay Taralp, Sabanci University
Protein purity and analysis
Q: What is PURE ENOUGH?
Q: What is the meaning of Purity? Free
of other proteins? Free of ions? Free of
DNA? Free of protease activity?
Q: In the case of insulin, what parameters should you analyze (and how)?
Size (many)
Primary sequence (many)
N-terminus, C-terminus (many)
Surface-accessibility of various groups (Chem. Mod.)
Shelf-life at different humidities (Solubility)
Amino group count (Kaiser test)
Histidyl group count (Pauli test)
pKa of ionizable groups (competative labeling)
Secondary sequence (CD)
Tertiary sequence (X-ray, NMR, receptor assay)
© 2006, Alpay Taralp, Sabanci University
e.g. Purifying insulin as a drug product
Routes of application:
Injection of suspended insulin under skin
Implant materials
Microcapsules containing insulin for lung absorption
What aspects of purity should you examine if you prepare
insulin as a drug formulation?
Sterility
Storage life and requirments
Biological activity
Bioavailability
Many of the previous criteria!
© 2006, Alpay Taralp, Sabanci University