protein structure. protein structure i primary structure
TRANSCRIPT
Protein Structure
Protein Structure I
Primary Structure
Figure 5-1
Primary Structure Insulin
Signal sequence Chain B
MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLV
C Peptide
CGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEG Chain A
SLQKRGIVEQCCTSICSLYQLENYCN
Bovine: Insulin
Human: ProInsulin
Figure 5-1
Primary Structure Insulin
Signal sequence Chain B
MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLV
C Peptide
CGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEG Chain A
SLQKRGIVEQCCTSICSLYQLENYCN
Bovine: Insulin
Human: ProInsulin
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
Figure 5-12
Sequencing
StrategySummary
Figure 5-12
Sequencing Strategy I
Figure 5-12
Sequencing Strategy II
Figure 5-12
Sequencing Strategy III
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
O2N F
NO2
O2N NH
NO2
CH
R
C
O
O2N NH
NO2
CH
R
COOH
H3N CH
R
C
O
DNFB(2,4 Dinitrof luorobenzene)
+
+ FreeAmino Acids
DNP- amino acid(Dinitrophenyl- amino acid)
H+ + HF
H+
Dansyl Chloride
NCH3 CH3
S
Cl
H2N C
H
R
C
NCH3 CH3
S OO
HN C
H
OO
O
R
C
O
Dansyl Chloride1-Dimethylaminonapthalene-
5-sulf onyl chloride
Dansyl amino acid + Free Amino Acids
+
Base
HCl
H+
End Group Analysis(How Many Different Subunits?)
C-Terminal Identification
Reduction
NH CH
R
COO NH CH
R
CH2OH
_
AminoAcids + Amino
Alcohol
LiBH4
H+
Hydrazinolysis
NH CH
R1
C NH
O
CH
R2
COO– H3N CH
R
C NHNH2
O
H3N CH COO–
R2
+
hydrazides
onlyunaffected aa
:NH2NH2
Cleavage of Disulfide Bonds
Oxidative Cleavage
– O3S CH 2 CH 2 SO3 –CH 2 S S CH 2
O
HCOOH
Cysteic Acid
(Perf ormic Acid)Cystine
A B
Problem(Oxidation of Methionine to Methionine
Sulfone)
CH2 CH2 S CH3
O
O
Reduction and Alkylation
CH2 S S CH2 CH2 SH HS CH2
"cystine"A B BA
2 R–SH R–S–S–R
-mercaptoethanol
HOCH2CH2SH
Problem
CH2 SH HS CH2 CH2 S S CH2"oxidation"
A—B, A—A, B—B
A B
O2
Solution
R SH + ICH2CONH2
"iodoacetamide"
HI
RSCH2CONH2
Separation and Purification of Polypeptide Chains
Sequence Polypeptide Chains
• Specific peptide cleavage reactions
• Separation and purification of peptide fragments
• Sequence determination
Hydrolysis
Polypeptide Amino AcidsHydrolysis
Acid Hydrolysis
I ndividual Amino Acids24 h
110o
Seal
Protein6N HCl
N2
Ala + Asp + Ser*110°, 24h6N HCle.g. AlaAspSer
Mechanism
H H
: :
H 3 N CH
R 1
C NH
O
CH
R 2COO
H 3 N CH
R 1
C NH
O
CH
R 2COO
H3N CH
R 1
COO–
H3N CH
R 2
COO–
H H
C NH
O
HH
+
+
+ _
_+
O H+O
H+
Problems
• Complete destruction of Trp
• Partial destruction of Ser, Thr, and Tyr
• Deamination of Asn and Gln
Deamination of Asn and Gln
R 1
H 3 N
C
C
N
C
C
N
C
C
O CH 2 H O
R 2OH
C
O NH 2
+
Asn
(also hydrolysis of peptide bonds)
H+
Base Hydrolysis(Many Amino Acids Destroyed)
(Racemization)
NH C
H
NH C
CH 3
NH C
CH 3
OO O
H B
D-amino acidL-amino acid
_
base + :BH
CC
CH3
C
B:
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
. . . NH CH
R 1
C NH
O
CH C . . .
R 2
O
Cleave peptide bonds
Specificity: R1
Table 5-3
Specificity of Endopeptidases
Chemical Cleavage(Cyanogen Bromide)
• • • NH CH C NH • • •
CH2
CH2
S CH3
C N
Br
• • • NH CH C NH • • •
OCH2
CH2 S CH3
C N
S CH3
C N• • • NH
CH C NH • • •
CH2 OCH2
• • • NH CH C O
CH2 OCH2
peptidyl homoserine lactone
+
peptide
+
+ NH3 • • •
methyl thiocyanate
+
..
H2O
Br
O
Separation and Purification of Peptide Fragments
Sequence Determination:
-Edman degradation-Mass Spectrometry
Edman Degradation I
NH C NH
S
C
H
R1
C NH
O
PTC (Phenylthiocarbamyl–)Polypeptide
N C S
Base
H2N C C
R 1
H
O
Phenylisothiocyanate
+
Edman Degradation II
NH C NH
S
C
H
R1
C NH
CH C
R2 O
O
NHS
NR1
O
ThiazolinoneDerivative
Anhydrous HF
PTC (Phenylthiocarbamyl–)Polypeptide
+ H3N Polypeptide
Edman Degradation III
NC
C
S
O
NH
CH R
CH C
R2 O
NHS
NR1
O
ThiazolinoneDerivative
PTH (Phenylthiohydantoin) Amino Acid
+
Mild Acid
H3N Polypeptide
Figure 5-16a part 1
Electrospray Ionization Mass Spectrometry (ESI)
Figure 5-16a part 2
Electrospray Ionization Mass Spectrometry (ESI)
Figure 5-16b
Electrospray Ionization Mass Spectrometry (ESI)
Figure 5-17
Tandem Mass Spectrometry
Organize Completed Structure
• Ordering peptide fragments
• Assignment of disulfide bond positions
• Determine position of amides
Ordering Peptide Fragments
Figure 5-18
Generating Overlapping Fragments
Ordering Peptide Fragments
Leu • Gly • Arg • Ala • Gly • Lys
Tyr • Lys • Glu • Met
Glu • Met • Leu • Gly • Arg
Tyr • LysTrypsin
CNBr
Overlapping Fragments
Tyr • Lys • Glu • Met • Leu • Gly • Arg • Ala • Gly • Lys
Ala • Gly • Lys
Complete Amino Acid Sequence
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
Table 5-5 part 1
Cytochrome c
All look like this
Sequence Comparisons Provide Information on Protein Structure
and Function
• Homologous proteins: evolutionarily related proteins
– Invariant residues
– Conservative substitutions
– Hypervariable positions
• Neutral drift
Figure 5-21
Phylogenetic Trees Depict Evolutionary History
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
Figure 5-22
Globin Family
Figure 5-23
The Rate of Sequence Divergence Varies