Protein Structure
Protein Structure I
Primary Structure
Primary Structure Insulin
Bovine: Insulin
Figure 5-1
Human: ProInsulin
Signal sequence
Chain B
MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLV
C Peptide
CGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEG
Chain A
SLQKRGIVEQCCTSICSLYQLENYCN
Primary Structure Insulin
Bovine: Insulin
Figure 5-1
Human: ProInsulin
Signal sequence
Chain B
MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLV
C Peptide
CGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEG
Chain A
SLQKRGIVEQCCTSICSLYQLENYCN
Value of Primary Structure Information
• Primary sequence information is
– prerequisite for determining three-dimensional structure
– essential in understanding molecular mechanism of action
• Sequence comparisons among analogous proteins
– provide insights into protein function
– reveal evolutionary relationships
• Sequence of proteins whose mutations result in inherited
diseases
– assist in development of diagnostic tests
– assist in development of effective therapies
Primary Structure Determination
Strategy
• Purification of protein to homogeneity
• Prepare protein for sequencing
• Sequence polypeptide chains
• Organize completed structure
Alternative: Nucleic Acid Sequencing
Sequencing
Strategy
Summary
Figure 5-12
Sequencing Strategy I
Figure 5-12
Sequencing Strategy II
Figure 5-12
Sequencing Strategy III
Figure 5-12
Purification of Protein to
Homogeneity
Prepare Protein for Sequencing
• End Group Analysis: How many different
subunits
• Cleavage of disulfide bonds
• Separation and purification of the
polypeptide chains
• Amino acid composition
End Group Analysis
(How Many Different Subunits?)
N-Terminal Identification
Sanger’s Reagent
H+ + HF
NO2
R
+
O2N
F
H3N
NO2
O
CH C
O2N
DNFB
(2,4 Dinitrofluorobenzene)
R
NH CH C
H+
NO2
O2N
NH
R
CH
DNP-amino acid
(Dinitrophenyl-amino acid)
COOH
+
O
Free
Amino Acids
Dansyl Chloride
CH3
CH3
CH3
CH3
N
N
+
O
S
H2N
R
O
C
C
H
O
HCl
Base
O
S
Cl
Dansyl Chloride
1-Dimethylaminonapthalene5-sulfonyl chloride
H+
Dansyl amino acid + Free Amino Acids
HN
O
R
O
C
C
H
End Group Analysis
(How Many Different Subunits?)
C-Terminal Identification
Reduction
R
NH CH
_
COO
LiBH4
R
NH CH
CH2OH
H+
Amino
Acids +
Amino
Alcohol
Hydrazinolysis
R1
NH CH
O
C
R2
NH
CH
COO–
H3N
R
O
CH
C
NHNH2
hydrazides
:NH2NH2
+
R2
H3N
CH
COO–
only
unaffected aa
Cleavage of Disulfide Bonds
Oxidative Cleavage
O
HCOOH
CH 2
S
S
CH 2
(Performic Acid)
A
Cystine
B
CH 2
SO3 –
–
O3S
Cysteic Acid
CH 2
Problem
(Oxidation of Methionine to Methionine Sulfone)
O
CH2
CH2 S CH3
O
Reduction and Alkylation
2 R–SH
CH2
A
S
S
"cystine"
R–S–S–R
CH2
CH2
A
B
HOCH2CH2SH
-mercaptoethanol
SH HS CH2
B
Problem
O2
CH2 SH HS CH2
A
CH2
"oxidation"
S
S
CH2
B
A—B,
A—A,
B—B
Solution
HI
R SH
+ ICH2CONH2
"iodoacetamide"
RSCH2CONH2
Separation and Purification of
Polypeptide Chains
Sequence Polypeptide Chains
• Specific peptide cleavage reactions
• Separation and purification of peptide
fragments
• Sequence determination
Hydrolysis
Polypeptide
Hydrolysis
Amino Acids
Acid Hydrolysis
Seal
N2
Protein
6N HCl
e.g. AlaAspSer
110o
24 h
6N HCl
110°, 24h
Individual Amino Acids
Ala + Asp + Ser*
Mechanism
+
H3N
R1
CH
R2
O
C
NH
CH
_
COO
H+
H
+
R1
H3N
CH
O
C
+
R2
NH
CH
H
_
COO
O
C
: :
O
H
NH
H+
O
H
H
R1
H
H3N
CH
COO–
+
R2
H3N
CH
COO–
Problems
• Complete destruction of Trp
• Partial destruction of Ser, Thr, and Tyr
• Deamination of Asn and Gln
Deamination of Asn and Gln
NH 2
O
C
+
H3N
O
CH 2
H
O
C
C
N
C
C
N
C
C
R1
H
O
R2
Asn
H+
(also hydrolysis of
peptide bonds)
Base Hydrolysis
(Many Amino Acids Destroyed)
(Racemization)
B:
H
CH3
C
NH
C
O
L-amino acid
CH 3
base
H B
CH 3
C
NH
H
+ :B
C
C
O_
NH
C
O
D-amino acid
Enzymatic Hydrolysis
Mild Conditions
Many proteases and peptidases
Specific and non-specific
Problem: contribution of amino
acids from hydrolysis of proteases
Amino Acid Analysis
(Automated)
Ion-exchange chromatography
High performance liquid chromatography
Colorimetric Analysis
Specific Peptide Cleavage Reactions
Proteolytic Enzymes
R1
. . . NH
CH C
O
R2
NH CH C . . .
O
Cleave peptide bonds
Specificity: R1
Specificity of Endopeptidases
Table 5-3
Chemical Cleavage
(Cyanogen Bromide)
• • • NH
CH
C
NH • • •
• • • NH
CH2 O
C
C
CH2 O
CH2
CH2
S
CH
CH3
N
Br
• • • NH CH
CH2
• • • NH
C
+
O + NH3 • • •
CH2
peptidyl homoserine
lactone
peptide
+
S CH3
C
N
S
Br
O
NH
.. • • •
H2 O
+
CH C NH • • •
CH2
O
CH2
C
CH3
N
methyl thiocyanate
Separation and Purification of
Peptide Fragments
Sequence Determination:
-Edman degradation
-Mass Spectrometry
Edman Degradation I
R1 O
N
C S
+ H2N
C C
H
Phenylisothiocyanate
Base
R1 O
NH
C NH C C NH
S
H
PTC (Phenylthiocarbamyl–)
Polypeptide
Edman Degradation II
R1 O
NH
C NH C C NH
H
S
PTC (Phenylthiocarbamyl–)
Polypeptide
Anhydrous HF
N
R1
R2
+
NH
S
H3N CH
O
Thiazolinone
Derivative
O
C
Polypeptide
Edman Degradation III
R1
N
R2
+
NH
S
H3N CH
O
Thiazolinone
Derivative
Mild Acid
S
C
NH
N
C CH R
O
PTH (Phenylthiohydantoin) Amino Acid
O
C
Polypeptide
Electrospray Ionization Mass
Spectrometry (ESI)
Figure 5-16a part 1
Electrospray Ionization Mass
Spectrometry (ESI)
Figure 5-16a part 2
Electrospray Ionization Mass
Spectrometry (ESI)
Figure 5-16b
Tandem Mass Spectrometry
Figure 5-17
Organize Completed Structure
• Ordering peptide fragments
• Assignment of disulfide bond positions
• Determine position of amides
Ordering Peptide Fragments
Generating Overlapping Fragments
Figure 5-18
Ordering Peptide Fragments
Overlapping Fragments
Trypsin
Tyr • Lys
Glu • Met • Leu • Gly • Arg
Ala • Gly • Lys
CNBr
Tyr • Lys • Glu • Met
Leu • Gly • Arg • Ala • Gly • Lys
Complete Amino Acid Sequence
Tyr • Lys • Glu • Met • Leu • Gly • Arg • Ala • Gly • Lys
Assignment of Disulfide Bond
Positions
Hydrolyze without breaking disulfides
Reduce, alkylate, and identify linked
fragments (disulfides)
Assignment of Amide Positions
Hydrolyze without breaking amides
Hydrolyze fragments and measure NH3
(need fragments having a single Asn or Gln)
Protein Evolution
Evolution by Natural Selection
Mutations
Cytochrome c
All look like this
Table 5-5 part 1
Sequence Comparisons Provide Information
on Protein Structure and Function
• Homologous proteins: evolutionarily related
proteins
– Invariant residues
– Conservative substitutions
– Hypervariable positions
• Neutral drift
Phylogenetic Trees Depict
Evolutionary History
Figure 5-21
Proteins Evolve by the Duplication
of Genes or Gene Segments
Protein Families Can Arise
through Gene Duplication
• Orthologous proteins: homologous proteins
with the same function in different species
• Paralogous proteins: independently evolving
proteins derived by duplication of a gene
(globin family)
• Pseudogenes
Globin Family
Figure 5-22
The Rate of Sequence Divergence Varies
Figure 5-23