Download Center for Structural Biology

Review: Amino Acid Side Chains
Aliphatic- Ala, Val, Leu, Ile, Gly
Polar- Ser, Thr, Cys, Met, [Tyr, Trp]
Acidic (and conjugate amide)- Asp, Asn, Glu, Gln
Basic- Lys, Arg, His
Aromatic- Phe, Tyr, Trp, [His]
Proline
R
a
N
vs
H
N
a
Review: Backbone Conformation
f
R
H

Ca
Ca
H
R
Ca
H
R
 Side chains collision also limit f/ combinations
 Backbone restricted  Secondary structure limited
Review: Heirarchy of Structure
Primary- sequence
Secondary- local
Supersecondary (motifs)- intermediate
Domains- independent folding units
Tertiary- organization of a complete chain
Quaternary- organization of multiple chains
Review: Tertiary Structure
Soluble proteins have an inside (core) and outside
 Folding driven by water- hydrophilic/phobic
 Side chain properties specify core/exterior
 Some interactions inside, others outside
Specific structures result from side chain interactions
Hydrophobic interactions (interior)
Hydrogen bonds (interior and exterior)
Ionic Interactions (exterior)
Relationships Among Proteins
Many sequences can give same tertiary structure
 Side chain pattern more important than
sequence
When sequence homology is high (>50%), probably
same structure and function (structural genomics)
 Cores conserved
 Surfaces and loops more variable
*3-D shape more conserved than sequence*
*There are a limited number of structural frameworks*
Relationships Among Proteins
I. Homologous: conserved sequence (cytochrome c)
 Same structure
 Same function
 Modeling structure from homology
II. Similar function- different sequence (dehydrogenases)
 One domain same structure
 One domain different
III. Similar structure- different function (cf. thioredoxin)
 Same 3-D structure
 Not same function
How to Tell Proteins Apart!
Sequence and fold give overall properties
Molecular weight
Solubility
Exposed hydrophobic surface
Ability to bind other molecules, metals
pI- the overall charge of the protein
Sequence!!!
*To characterize properties, separate the
protein from all other cell contents*
Protein Purification Techniques
A. Simple solubility characteristics- precipitation
 Temperature
 pH
 “Salting out”
*Different proteins precipitate under different solution
conditions- can use soluble or insoluble fractions*
Protein Purification Techniques
B. Chromatography- fractionation of contents in
solution based on selection by a stationary phase
1. Size- sieve effect, small molecules faster
2. Ion exchange- charge attraction at protein
surface
 Choose “+” stationary phase for proteins with
more “-” charge
 First bind everything, then elute with salt
3. Hydrophobic interaction- hydrophobic
accessible surface
4. Affinity chromatography
 Antibody, binding protein
 Inserted tag (e.g. 6-His)
Protein Purification Techniques
C. Gel Electrophoresis- migration in a gel matrix (size
and shape) driven by an electric field (charge)
 Sieving effect
 Relative charge
 Visualization- staining with dye, fluorescent
antibody (Western blotting)
 SDS- protein denaturant, enables separation
based almost exclusively on molecular weight
 Iso-electric focusing- method to measure pI, but
also can be used for separation
Chromatography and SDS-PAGE
2000
(Lanes 1, 2)
1800
M1234 56789
(Lanes 3, 4)
1600
I
1400
54.4
36.5
1200
1000
21.5
14.4
800
(Lanes 7, 8, 9)
600
400
200
0
0
20
40
60
80
Volume (ml)
100
120
140
Fusion
protein
GST
T-ag
Protein Characterization
A. Sequence
1. Amino acid analysis- total digest, then count how
much of each amino acid
2. Edman stepwise degradation- cleave of one
residue at a time, then identify
3. Peptide mapping- cleave into fragments, then
identify
4. Direct sequencing by Mass Spectrometry
 Exact molecular weights
 Characteristic fragmentation
Protein Characterization
B. Spectroscopic properties
1. UV-Vis- Backbone, Phe, Tyr, Trp, co-factors
2. Infrared/Raman- characteristic bond vibrations
3. Circular Dichroism (CD)- backbone conformation
4. Fluorescence
 Intrinsic- Trp, Tyr
 Attached dyes- Cys
5. Electron Paramagnetic Resonance (EPR)
 Metals, free radicals
 Attached probes
6. Nuclear Magnetic Resonance (NMR)
 Many probes viewed simultaneously
 Structure and dynamic processes
Protein Characterization
C. Antibodies
 Use protein of interest to raise antibodies (rabbit)
 Different antibodies can recognize different
regions (epitopes)
 Can distinguish differences as small as 1 residue
 Attachment of indicators- dyes, radioactivity
 Applications- e.g. immunoassay, ELISA