Download Chapter 3 Amino Acids, Peptides and Proteins

Chapter 3
Amino Acids, Peptides
and Proteins
Amino Acids
• Amino acids can be formed from inorganic compounds
under prebiotic conditions
• These compounds are the monomeric units that are joined to
form polypeptides (aka. Proteins)
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1
Amino Acids
Structural Features
All proteins are built from a combination of the 20 amino acids
H C
H
α
R
C
pH
COOH
R
C
COO-
Carboxyl
Amino
NH3+
NH2
• “Amino” “acids” are built around a central “α-carbon” atom
• Because an acid and a base are both present, an amino acid can form
either a positive or negative ion
• In fact, at physiological pH, both the acid and the base are completely
ionized, resulting in a compound with both positive and negative charge
called a Zwitterion
Chapter 3
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Amino Acids
Structural Features
• Four different substituents on the α-carbon
atom means that the alpha carbon is a chiral
carbon (Enantiomers!!)
• These chiral centers are optically active,
meaning they rotate plane-polarized light
– Rotating light to the left – Levorotatory (L)
– Rotating light to the right – Dextrorotatory
(D)
• To determine whether your amino acid (or
sugar!) is D or L, you need to look at the
fischer diagram with the carboxyl at the top
and the R group on the bottom.
• If the amino group is to the LEFT then the
amino acid is “L”!
Chapter 3
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2
Amino Acids
Structural Features
• Asymmetry (D vs. L) and side chain differences result in great
variety in the polypeptides formed
• The amino acid residues in proteins are exclusively L
stereoisomers.
• There are other L-amino acids in living cells
– Some as biochemical intermediates
– Some with modified R-groups after synthesis
• D-amino acids have been found in only a small number of
peptides:
– The peptides of bacterial cell walls and in antibiotic peptides.
• Overall, ~ 300 different amino acids occur in living organisms!!
• Gives nature a lot to work with when putting together proteins!
Chapter 3
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Amino Acids
Classification of Amino Acids
All amino acids have the same core structure, but vary in their side chains
(the “R” group), which effects their physiochemical properties
H
R
C
COO-
NH3+
• The variation in the R group allows the amino acids to be grouped
based on the Polarity of their R group
• They fall into five distinct groupings as clusters of 7, 5, 3, 3, and 2:
the aliphatics, polars, aromatics, basics, and acidics
Hydrophobic “Water fearing” Non-polar side chains
Hydrophilic “Water Loving” Polar, neutral or charged side chains
Chapter 3
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3
Amino Acids
The Seven Aliphatics (Non-polar)
These seven amino
acids tend to cluster
together within proteins,
stabilizing structure
through hydrophobic
interactions.
Chapter 3
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Amino Acids
The Five Uncharged (Polar)
The R groups of these five
amino acids are more soluble
in water than those of the
alphatic AAs due to the
presence of hydroxyl, thiol
and amide groups.
Chapter 3
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4
Amino Acids
The Three Aromatics
These three amino acids are relatively non-polar and all can
participate in hydrophobic interactions. They all also absorb
UV light due to their conjugation. The hydroxyl group of Tyr
also allows this residue to form hydrogen bonds.
Chapter 3
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Amino Acids
The Three Basics
These three amino acids (along with the acidic AAs)
are the most hydrophilic AAs and can participate in
hydrogen bonding interactions as H-bond Donors.
Chapter 3
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5
Amino Acids
The Two Acidics
These two amino acids (along with the basic AAs)
are the most hydrophilic AAs and can participate in
hydrogen bonding interactions as H-bond acceptors.
Chapter 3
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Amino Acids
Other ways to Group the AAs
Which and how many…
• … have hydroxyl groups?
• … contain sulfur?
• … contain 5- or 6-membered rings?
• … have pure hydrocarbon side chains?
• … have nitrogen in their side chains?
• Which is biggest? …smallest?
…most acidic? …most basic?
Chapter 3
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6
Amino Acids
Chapter 3
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Amino Acids
Classification of Amino Acids
Chapter 3
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7
Amino Acids
Essential Vocabulary for CHE 421
• You will be required to learn the
structure, name, abbreviation, pKa values
(average ones for the carboxyl and
aminol; specific value for ionizable R
groups!) and unique characteristics of
each of these 20 amino acids.
• You will need command of these structures
to be successful in the rest of this course…
Chapter 3
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Amino Acids
Unusual L-Amino Acids Found in Proteins
• In addition to the 20
standard AAs, proteins
can (and do!) contain
residues formed from the
modification of common
residues already
incorporated into a
polypeptide.
• Selenocysteine is a rare
AA that is introduced into
the protein as the modified
version rather than
modified once in place.
Fig. 3-8a
Chapter 3
Sometimes referred to as the “21st Amino Acid”
16
8
Amino Acids
Unusual L-Amino Acids Found Elsewhere
• Not all AAs are found in polypeptides.
• Many are found in metabolic and synthetic pathways.
• In what synthetic (ornithine) and degradation (citrulline)
pathways do you think that ornithine and citrulline show up as
intermediates?
Fig. 3-8a
Chapter 3
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Amino Acids
The Amino Acid as Zwitterion
•
An AA has at least one acidic
(carboxyl) and one basic (amino)
group
– If both are charged at the same time,
the AA is a zwitterion
•
The R-group may also contribute
charge
•
Due to these multiple ionizable
groups, a titration curve for an AA
will have a minimum of two inflection
points
•
What happens to the structure
above and below the pKa values for
the amino and carboxyl groups?
– Y, C, K, H, R, D and E
Chapter 3
What form would be present
at physiological pH, 7.4?
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9
Amino Acids
Titration of an Amino Acid
• Where does the zwitterion
form occur?
• If the pKa of the carboxyl
group of acetic acid is 4.76,
why is the pK1 of glycine’s
carboxyl group so much
lower, at 2.34?
• How would the titration
curve of Histidine vary?
Chapter 3
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Amino Acids
Titration of a Charged Amino Acid
• Why is the pK1 of
Histidine’s carboxyl
group 1.82 compared to
2.34 for Glycine?
• How would the titration
curve of Lysine vary?
Chapter 3
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10
Amino Acids
Titration of an Amino Acid
Local Chemistry Affects the Charge Properties of Amino Acids
Chapter 3
pK1 lies between pH 1.8-2.4
pK2 lies between pH 8.8-11.0
21
Amino Acids
Isoelectric Point (pI)
• The characteristic pH at which the net electric charge of
the amino acid is zero is the isoelectric point (pI)
• For amino acids with a non-ionizable R group, this
value is equal to the average of the two pKa values:
pI = ½ (pK1 + pK2)
• At pH values above the pI what is the overall charge on
the amino acid?
• What about below the pI?
• Why would this be good info to know?
Chapter 3
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11
Amino Acids
Titration of a Charged Amino Acid
What about the pI
for AAs with an
ionizable R group?
Where’s the
Zwitterion?
Chapter 3
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Amino Acids
Titration of a Charged Amino Acid
Isoelectric Points for Amino Acids with Ionizable R-groups
• If an amino acid has two amino groups, or two
carboxyl groups, the pI is equal to the average of
the values of the two like groups.
• Example: L-Histidine
pK1 = 1.82, pK2 = 6.00, pK3 = 9.17
• Therefore:
pI = (6.00 + 9.17)/2 = 7.59
Why does this rule of thumb work?
Chapter 3
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12
Amino Acids
Can You Estimate the Number of
Amino Acids in a Protein?
• The answer is yes!
• First, you need to know that the weighted
average MW of the 20 standard amino acids
is 128.
• Next, you take the molecular weight of the
protein and divide it by 110 (WHY? What
happened to the other 18?)
– e.g. a protein of 55 kD = MW 55,000 is
comprised of about 500 amino acids…
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Amino Acids
Functional Groups in Macromolecules Can
Provide Buffering Capacity
Chapter 3
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13
Amino Acids
But Enzyme Catalysis is Also pH-sensitive
Why might
this be?
Think about it…
Chapter 3
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Amino Acids
Let’s See Who Learned Their AAs this Weekend
Amino Acid Quiz
http://www.biology.arizona.edu/biochemistry/problem_sets/aa/aa.html
Chapter 3
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Peptides and Proteins
Proteins Result from Peptide Bond Formation
(a Condensation Reaction)
Poor leaving group
The peptide bond is formed
through a nucleophilic
displacement that takes place
in the ribosome during
translation.
Good nucleophile
∆G = + 21 kJ/mole
(very unfavorable – so why
are proteins so stable?)
Chapter 3
Peptide bond
29
Peptides and Proteins
Can You Estimate the Isoelectric Point of a Protein?
• Review the “rule of thumb” for calculating the pI’s of
amino acids.
– How might this rule apply to peptides and proteins?
• Remember that polypeptides contain only one free αCOOH and α-NH2 group, at opposite ends of the chain
• The R groups of some of the AAs in the polypeptide will
be ionizable and contribute to the pI of the protein.
• Remember, the pKa values for both the termini and the
R groups are effected by the chemical environment, so
their use in this type of calculation will give you an
estimated pI only (better to determine in the lab!)
Chapter 3
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15
Peptides and Proteins
A Pentapeptide
Chapter 3
What is the AA sequence?
What is the approximate pI?
31
Peptides and Proteins
AA Composition of Proteins
• Each protein has a characteristic mix
of AAs in its sequence.
• No two proteins are identical in this
makeup.
• Some AAs appear frequently and other
not so much.
Chapter 3
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16
Working with
Proteins
pI and AA Composition
Chapter 3
Lysozyme
Pepsin A
Amino Acid
# in PP (%)
% in PP
Ala
12 (9.3%)
16 (4.8%)
Arg
3 (2.3%)
3 (0.9%)
Asn
11 (8.5%)
15 (4.5%)
Asp
5 (3.9%)
22 (6.7%)
Cys
8 (6.2%)
6 (1.8%)
Gln
5 (3.9%)
15 (4.5%)
Glu
2 (1.6%)
14 (4.2%)
Gly
12 (9.3%)
34 (10.3%)
His
0 (0.0%)
1 (0.3%)
Ile
5 (3.9%)
30 (9.1%)
21 (6.4%)
Leu
9 (7.0%)
Lys
17 (13.2%)
0 (0.0%)
Met
1 (0.8%)
3 (0.9%)
Phe
2 (1.6%)
15 (4.5%)
Pro
2 (1.6%)
15 (4.5%)
Ser
9 (7.0%)
49 (4.8%)
Thr
8 (6.2%)
26 (7.9%)
Trp
5 (3.9%)
5 (1.5%)
Tyr
5 (3.9%)
17 (5.2%)
Val
8 (6.2%)
23 (7.0%)
# of Negatively
Charged Residues
7
36
# of Positively
Charged Residues
20
3
pI
9.55
33
3.55
Peptides and Proteins
Multimeric Proteins
Average AA molecular
weight is 110.
We can use this value
to estimate the
number of AAs in a
protein if we know the
MW of that protein.
• Multisubunit proteins consist of two or more separate polypeptide chains.
• If at least two of these chains are identical, the protein is oligomeric and
each chain is a protomer
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Peptides and Proteins
Conjugated Proteins
• In addition to AAs, many
proteins contain permenantly
associated chemical groups
called prosthetic groups
• These groups are often
involved in the catalytic
activity of enzymes or in
structural maintainence
• Conjugated proteins are
classified on the basis of the
attached prosthetic group.
• Proteins can also contain
more than one of these
groups
Chapter 3
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Peptides and Proteins
Protein Structure Beyond the AA Sequence
CHP 3.4
Chapter 3
CHP 4
36
18
Working with Proteins
Separation and Purification of Proteins
• A pure sample of protein is vital to determining its structure and function
• Proteins can be separated by exploiting their differences which are determined by their
structure and amino acid sequence
Solubility
Ammonium Sulfate Precipitation
Crystallization
Charge
Ion Exchange Chromatography
Isoelectric Focusing
Size
Gel Filtration Chromatography
SDS-PAGE Electrophoresis, Dialysis
Specific Binding
Special Properties
Chapter 3
Affinity Chromatography
Heat Denaturation
Engineered Properties
37
Working with Proteins
Separation and Purification of Proteins
Cell Lysis
Soluble Fraction
Centrifugation to remove
cell debris/insoluble
components
Liquid
chromatography
Removal of
nucleic acids
(Optional)
Protein Fraction
Crude fractionation
by precipitation
Purified
protein
Chapter 3
Liquid
chromatography
Enriched
protein fraction
38
19
Working with Proteins
Column Chromatography
• In any type of column
chromatography, you have the
mobile phase and the stationary
phase
• There are two types of stationary
phases:
– Non-adsorptive: No interaction
between the solute and the SP
– Adsorptive: Chemical interaction
between the solute and the SP. You
must alter your MP to elute the solute.
• As with all chromatography, you
must equilibrate you column with MP
prior to loading and eluting your
solute!
Chapter 3
39
Working with Proteins
Column Chromatography – Ion Exchange (IEC)
• Proteins differ in the number of charged amino
acids (Asp, Glu, His, Arg, and Lys) found in their
AA sequence.
• Therefore, they will vary in their overall charge at
various pH values and the pH value at which their
net charge is zero (their isoelectric point, pI)
• In IEC, the SP contains charged groups: Anion
exchange (+++) / Cation exchange (- - - )
• Strong and weak ion exchangers are available
depending on your protein
• Sample eluted with increasing salt or change in pH
• Isoelectric point of protein generally needs to be
known or several pilot runs need to be performed
Chapter 3
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20
Working with Proteins
Column Chromatography – Size Exclusion (SEC)
• A protein’s size and shape affect its ability to
move through a matrix of of porous beads or a
porous membrane
• Proteins elute in decreasing order of size
• The SP is a non-absorptive & porous matrix
• SP with varying pore sizes are available
• Some uses include: Separating contaminating
solutes and Protein fractionation
Chapter 3
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Working with Proteins
Column Chromatography – Affinity (Aff)
• Proteins can naturally or artificially
contain special binding properties:
– Receptor-ligand interactions
– Protein-protein interactions
• Samples eluted by various means
dependent on the binding interaction
• The SP contains specific ligands
dependent on the binding property being
exploited:
– Dyes
– Metals
– Substrates and cofactors
• Aff has a very high selectivity that often
yields pure or nearly pure protein in
minimal steps.
Chapter 3
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21
Working with Proteins
Electrophoresis
• Electrophoresis is a method of
molecule separation that exploits the
size and charge of the components
• Here, the application of an electrical
field to macromolecules in solution will
cause them to migrate to either the:
– + anode (negatively charged molecules)
– cathode (positively charged molecules)
• Gels typically used as a support matrix
act as molecular sieves through which
molecules may or may not be able to
pass based on their size:
– Agarose (DNA) and Polyacrylamide
(Proteins)
Chapter 3
43
Working with Proteins
Electrophoresis
•
SDS (sodium dodecyl sulfate) binds to proteins via
hydrophobic interactions
– Usually about 1 SDS per 2 AAs
•
•
When SDS added in excess:
– proteins “coated” with SDS, thus “masking”
charge of protein and imparting overall negative
charge
– proteins are denatured, meaning organizational
structure of molecule is disrupted
To completely denature protein, add a reducing
agent such as β-Mercaptoethanol (BME),
dithiothreitol (DTT) and/or heat
SDS-PAGE
Chapter 3
O
Na+ -
O
S
O
(CH2)11CH3
O
Sodium dodecyl sulfate (SDS)
44
22
Working with Proteins
Electrophoresis – Determination of MW
Chapter 3
45
Working with Proteins
Isoelectric Focusing
•
•
•
Procedure used to determine the pI of a
protein
Here, a pH gradient is established by
allowing a mixture of low MW organic
acids and bases distribute themselves in
an electric field generated across a gel.
When a protein mixture is added, the
protein components migrate until they
reach a pH that matches their pI value.
Let’s try Cytochrome c Peroxidase
It contains 295 AA’s
pI / Molecular Mass Calculator
Chapter 3
46
23
Working with Proteins
2-D Electrophoresis
•
•
•
2-D electrophoresis combines SDS-PAGE and IE
into one experiment.
This allows for the separation of proteins based on
both MW and pI.
Let’s us separate proteins that have the same pI
but varied MW and proteins that have the same
MW but varied pI.
Chapter 3
47
Working with Proteins
Enzyme Assay
• Once we purify out a protein or even during the purification itself, how do
we know we are focused in on our protein of interest? How do we know
we have not thrown it out with our last step?
• For enzymes, we can use the reaction that it catalyzes to monitor the
presence and amount of the enzyme throughout the purification
• To monitor this reaction, we need to design an assay that allows us to
observe the catalytic effect of our enzyme.
• To develop an enzyme assay we must know the following:
– The overall equation for the catalyzed reaction
– An analytical procedure for determining the disappearance of substrate or appearance
of product for the reaction
– Whether the enzyme requires co-factors such as metals or coenzymes for activity
– The dependence of the enzyme activity on substrate concentration
– The optimum pH for activity
– A temperature zone in which the enzyme is stable and has high activity
Chapter 3
48
24
Working with Proteins
Enzyme Activity
• One unit (1 U) of enzyme activity is defined
as the amount of enzyme causing the
transformation of 1.0 µmol of substrate per
minute at 25°C.
•
•
Activity is the total units of enzyme in a solution
Specific Activity is the number of enzyme units
per milligram of total protein.
Activity
Specific
Activity
Chapter 3
49
Covalent Structure of Proteins
Protein Sequence
• The AA sequence of a protein represents very
important information in determining its structure and
function
• A human produces 25000 to 35000 proteins, each of
which has a unique structure and sequence
• The AA sequence is important to the 3-D structure of
the protein, which in turn, is important to the function of
the protein.
• By knowing the AA sequence, we can make predictions
about the 3-D structure, and the function of the protein.
• In addition, by comparing AA sequences for the same
protein across species and within species, we can
identify evolutionary changes and genetic defects
which may cause disease
Chapter 3
50
25
Covalent Structure of Proteins
Protein Sequencing – AA Composition Analysis
•
•
•
•
•
•
•
In some cases, you may want to know the
number of each type of amino acid within a
polypeptide.
The AA composition can be determined by
completely hydrolyzing a protein under acidic
conditions then analyzing the AAs using
chromatography (usually via a detectable tag)
Different amino acids in a peptide hydrolysate
can be separated by ion-exchange
chromatography on a sulfonated polystyrene
resin (such as Dowex-50)
Figure 4.18 from Biochemistry, 5th Ed., Berg et al.
Buffers (such as sodium citrate) of increasing pH are used to elute the amino acids
from the column.
The amount of each amino acid present is determined from the absorbance.
Aspartate, which has an acidic side chain, is first to emerge, whereas arginine, which
has a basic side chain, is the last.
In this case, the original peptide is revealed to be composed of one aspartate, one
alanine, one phenylalanine, one arginine, and two glycine residues
Chapter 3
51
Covalent Structure of Proteins
Protein Sequencing
• Various procedures are available for determination of a protein’s amino
acid sequence.
– The Sanger Method
– The Edman Degradation Method
• Regardless of the technique used, the basic approach for sequencing
proteins is similar:
– Separate the mature protein into individual polypeptide strands (if needed)
– Use at least two different chemical and/or enzymatic methods to break the
polypeptide into two sets of smaller peptide segments
– Determine the sequence of each segment
– Reconstruct the overall sequence by ordering overlapping segments from
the two sets
– Repeat the fragmentation step without breaking intra-strand connections to
identify any Cys residues involved in disulfide bridges
Chapter 3
52
26
Covalent Structure of Proteins
Protein Sequencing – Preliminary Steps
•
•
•
The complete AA sequence of a protein includes the sequence of each of its subunits (if
any), so these subunits must be identified and isolated
Disulfide bonds within the protein structure must also be broken top separate and fully
lineraize polypeptide chains
Disulfide bonds can be reductively cleaved by treating them with 2-betamercaptoethanol
(BME) or another mercaptan (contains a –SH group)
O
H
N
H
C
O
H
N
C
CH2
S
SH
+
SH
+ 2 HSCH2CH2OH
•
C
SCH2CH2OH
+
SCH2CH2OH
CH2 O
CH2 O
C
H
C
CH2
BME
S
H
N
H
C
H
N
C
H
C
The resulting free sulfhydral groups are then alkylated, usually by treatment with
iodoacetate, to prevent reformation of the disulfide bond through O2 oxidation
CYS
Chapter 3
H2
C
SH + ICH2COO-
CYS
H2
C
S
CH2COO- + HI
53
Covalent Structure of Proteins
Protein Sequencing – Preliminary Steps
•
•
Once the disulfide bonds are disrupted, the subunits are allowed to separate (if
present) and the polypeptide(s) are linearized
Now, N-terminal analysis can reveal the number of different subunits.
– Frederick Sanger developed the reagent 1-fluoro-2,4-dinitrobenzene (FDNB) for this
purpose
– Other reagents include Dansyl chloride and Dabsyl chloride
– All of these reagents react with primary amines (the N-terminal amino group)
•
•
For this procedure, the reagent is added to the protein solution resulting in a
labeling of all amino terminii.
The labeled polypeptide(s) are then hydrolyzed into individual AAs and the label
is identified chromatographically.
H3C
CH3
N
Dansyl Chloride
SO2Cl
H3C
N
Chapter 3
N
N
SO2Cl
54
H3C
Dabsyl Chloride
27
Covalent Structure of Proteins
Protein Sequencing – Edman Degradation
•
•
•
If the polypeptide is small ( 50 AAs or less) you can determine the sequence
using repeated cycles of Edman Degradation
Here, Phenylisothiocyanate (PITC) reacts with the N-terminal amino group
under alkaline conditions to form a phenylthiocarbamoyl (PTC) adduct.
The peptide bond adjacent to this labeled terminus is cleaved under acidic
conditions (Thifluoroacetic acid)
– This treatment cleaves only the peptide bond of interest!
•
This derivative is then extracted in organic solvent and identified
Chapter 3
55
Figure 4.21 from Biochemistry,
5th Ed., Berg et al.
Covalent Structure of Proteins
Protein Sequencing – Polypeptide Cleavage
•
•
If the polypeptide is larger than ~ 50 AAs, it
must be cleaved, either chemically or
enzymatically, into specific fragments that
are small enough to be sequenced using
Edman Degradation.
Endopeptidases are enzymes that catalyze
the hydrolysis of internal peptide bonds
– These enzymes are in the enzyme family of
proteases and have side chain requirements
for the residues flanking the target peptide
bond (aka the scissle bond)
•
These fragments are then separated using
chromatography or electrophoresis
Figure 4.24 from Biochemistry, 5th Ed., Berg et al.
Chapter 3
56
28
Covalent Structure of Proteins
Protein Sequencing – Polypeptide Cleavage
H3N – NMTQGRCKPVNTFVHEPLVDVQNVCFK – COOYou must cleave the above
peptide into smaller fragments.
• Which of the proteases listed
would yield the most fragments?
• How many?
• Which would yield the least
fragments?
• How many?
Chapter 3
57
Covalent Structure of Proteins
Protein Sequencing – Ordering Peptide Fragments
•
•
•
•
Now that each fragment has been sequenced, the order of these fragments
must be determined so that the entire polypeptide sequence can be established
The AA sequence of the fragments formed from two separate cleavage
methods are examined to identify overlapping sequences.
Overlapping peptides can be identified and put in order
Earlier identification of the Amino terminus can be used to identify the amino
terminus fragment
Chapter 3
58
29
Covalent Structure of Proteins
Protein Sequencing – Ordering Peptide Fragments
• You wish to determine the sequence of a short
polypeptide.
• Cleavage with trypsin yields three smaller peptide:
L–E
G–Y–N–R
Q–A–F–V–K
• Cleavage with chymotrypsin yields three peptides:
Q–A–F
N–R–L–E
V–K–G–Y
What is the sequence of this polypeptide?
Chapter 3
59
Covalent Structure of Proteins
Protein Sequencing – Locating Disulfide Bonds
• If the primary sequence includes disulfide bonds, their location is also of
interest
• To determine the location of the disulfide bonds:
– Take a new sample of the protein and separate the polypeptides but DO
NOT reduce the disulfide bridges
– Subject this strand to the same chemical or enzymatic cleavage technique
as before and separate the fragments using electrophoresis
– Compare this pattern to that of the original sample
If there is one disulfide bridge in your polypeptide, what would
you expect the difference to be between the two samples?
How would you identify the location of the disulfide bridge?
Chapter 3
60
30
Covalent Structure of Proteins
Protein Sequencing – Locating Disulfide Bonds
• Treatment of a polypeptide with 2-mercaptoethanol yields two
polypeptides:
A–V–C–R–T–G–C–K–N–F–L
Y–K–C–F–R–H–T–K–C–S
• Treatment of the intact polypeptide with trypsin yields fragments with
the following AA composition
A, R, C2, S, V
R, C2, G, K, T, F
N, L, F
H, K, T
K, T
What are the positions of the disulfide bond(s)?
Chapter 3
61
Covalent Structure of Proteins
Protein Sequencing
Chapter 3
62
31
Protein Sequence and Evolution
Protein Sequencing and Mass Spectrometry
• Mass spectrometry has only recently
become a tool of the biochemist
– This technique accurately measures the
mass-to-charge ratio for ions in the gas
phase
• New ionization techniques have allowed for
the formation of macromolecule ions
without degradation of the compound
– Matrix-assisted laser desorption/ionization
(MALDI)
– Electrospray ionization (ESI)
• MS can be used to determined the
molecular mass of a protein and to detect
changes in this mass due to the presence
of cofactors, metal ions, covalent
modification due to reactions, etc.
• Tandem MS (MS/MS) can also be used to
sequence short stretches of polypeptide.
Chapter 3
63
Covalent Structure of Proteins
Protein Synthesis
•
Peptides are potentially useful as
pharmacological agents making their
production commercially profitable
•
There are three ways to produce a
peptide:
•
–
Purification from the biological source
–
Genetic engineering
–
Direct chemical synthesis
R. Bruce Merrifield developed an
innovative technique for peptide
synthesis by attaching a growing
polypeptide to an insoluble support and
blocking the growing end
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Protein Sequence and Evolution
Protein Sequence and Evolution
• As stated before, the function of a protein depends on its 3-D
structure, which, in turn, depends on its AA sequence
• The field of Molecular Evolution uses nucleotide and peptide
sequences to explore evolution
• Basically, if two organisms are closely related, the sequences of
their genes and proteins should be similar
• As organisms diverge evolutionarily, their sequences will also
diverge
• We now have the computer power to store, translate and
compare thousands of sequences, both peptide and nucleotide!
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65
Protein Sequence and Evolution
Databases Containing Gene and Protein Info
• After a protein’s AA sequence has been determined, the information is
usually deposited into a public database
• Several of these databases are available via the Internet and can be
used to determine valuable information regarding proteins, nucleic
acids and evolution
Data Banks Containing Protein Sequences
Swiss-Prot: http://au.expasy.org
Protein Info Resource: http://pir.georgetown.edu
Protein Research Foundation: http://www4.prf.or.jp
UniProt: http://www.ebi.uniprot.org/
Data Banks Containing Gene Sequences
GenBank: http://www.ncbi.nlm.nih.gov
European Bioinfor Institute: http://srs.ebi.ac.uk
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Protein Sequence and Evolution
Protein Sequence and Evolution
• There are some issues with this type of comparison, in that it is not as
straight forward as just lining things up and calling it a match
– First, the AA residues required for function will usually be conserved across
species while those residues of less importance may vary over time
– Second, there can be a rare transfer of a gene or group of genes from one
species to another (lateral gene transfer). This may result in a false
positive when running the sequence comparison. Can anyone think of an
example of this lateral gene transfer?
• The study of molecular evolution generally focuses on families of
closely related proteins, usually groups with functions that are essential
to the viability of the organisms
• When comparing peptide sequences, you can find homologs, which
can be either paralogs (in the same species) or orthologs (in different
species).
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Protein Sequence and Evolution
Protein Sequence and Evolution
•
A Blosum62 table (blocks substitution matrix complied from alignment that
showed 62% identity) shows the scores assigned to AA substitutions within
an alignment
•
Remember that gaps in the sequence alignments incur penalties as well
– These substitutions can be conservative or nonconservative
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Protein Sequence and Evolution
Protein Sequence and Evolution
• In site directed mutagenesis experiments, Gly is often
successfully substituted for Val, but Val can rarely substitute
for Gly. Why?
• Below is a list of the first 10 residues of the B helix in
Myoglobin from different species:
Position
1
2
3
4
5
6
7
8
9
Human
D
I
P
G
H
G
Q
E
V
10
L
Chicken
D
I
A
G
H
G
H
E
V
L
Alligator
K
L
P
E
H
G
H
E
V
I
Turtle
D
L
S
A
H
G
Q
E
V
I
Tuna
D
Y
T
T
M
G
G
L
V
L
Carp
D
F
E
G
T
G
G
E
V
L
Which positions appear unable to tolerate substitutions?
Which can tolerate conservative substitutions?
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Which are highly variable?
Protein Sequence and Evolution
Protein Sequence and Evolution
Tree based on
sequence
divergence in the
protein GroEL in
bacteria
•
The free end points of lines are external nodes and represent an extant species
•
The points where two lines come together are internal nodes and represent extinct
ancestor species
•
The lengths of the lines connecting the nodes are proportional to the number of amino
acid substitutions separating one species from another
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