Protein Structure. Protein Structure I Primary Structure.

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

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


Figure 5-16b


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

Protein Structure. Protein Structure I Primary Structure.

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