05. Protein Sequencing

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Copyright © 1998 W.R. MiddenAll Rights Reserved Bowling Green State University Protein Sequencing

How To Sequence A Protein

W. Robert Midden

Department of Chemistry

Bowling Green State University

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Protein SequencingPreliminary Steps

For multisubunit proteins, the individual protein chains must first be separated

Break interchain disulfide bonds, if necessary

Two reagents are commonly used: performic acid mercaptoethanol

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2-Mercaptoethanol

2-mercaptoethanol reduces disulfides to sulfhydryls But the sulfhydryls are easily oxidized back to the disulfide

S

S

C

H

2

C

H

2

C

H

C

H

N

H

C

C

N

H

C

C

O

O

O

O

S

H

S

H

C

H

2

C

H

2

C

H

C

H

N

H

C

C

N

H

C

C

O

O

O

O

S

H

C

H

2

C

H

2

O

H

S

C

H

2

C

H

2

O

H

S

C

H

2

C

H

2

O

H

S

H

C

H

2

C

H

2

O

H

S

S

C

H

2

C

H

2

C

H

C

H

N

H

C

C

N

H

C

C

O

O

O

O

S

H

S

H

C

H

2

C

H

2

C

H

C

H

N

H

C

C

N

H

C

C

O

O

O

O

S

H

C

H

2

C

H

2

O

H

S

C

H

2

C

H

2

O

H

S

C

H

2

C

H

2

O

H

S

H

C

H

2

C

H

2

O

H

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Preventing Reversal

to prevent oxidation the suflhydryls are alkylated with iodoacetic acid or acrylonitrile

S

H

C

H

2

C

H

N

H

C

C

O

O

S

C

H

2

C

H

N

H

C

C

O

O

C

H

2

C

H

C

N

C

H

2

C

H

2

C

N

S

H

C

H

2

C

H

N

H

C

C

O

O

S

C

H

2

C

H

N

H

C

C

O

O

C

H

2

C

H

C

N

C

H

2

C

H

2

C

N

S

H

C

H

2

C

H

N

H

C

C

O

O

I

C

H

2

C

O

O

S

C

H

2

C

H

N

H

C

C

O

O

C

H

2

C

O

O

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H

C

H

2

C

H

N

H

C

C

O

O

I

C

H

2

C

O

O

S

C

H

2

C

H

N

H

C

C

O

O

C

H

2

C

O

O

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Perfomic Acid

H

C

O

O

O

H

S

S

C

H

2

C

H

2

C

H

C

H

N

H

C

C

N

H

C

C

O

O

O

O

S

O

3

-

S

O

3

-

C

H

2

C

H

2

C

H

C

H

N

H

C

C

N

H

C

C

O

O

O

O

H

C

O

O

O

H

S

S

C

H

2

C

H

2

C

H

C

H

N

H

C

C

N

H

C

C

O

O

O

O

S

O

3

-

S

O

3

-

C

H

2

C

H

2

C

H

C

H

N

H

C

C

N

H

C

C

O

O

O

O

Performic acid oxidizes cysteine to negatively charged cysteic acid

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Reversal Prevented

The repulsion of the negatively charged SO3-

groups prevents reformation of the disulfide bond Therefore alkylation is not necessary with

performic acid

S

O

3

-

S

O

3

-

C

H

2

C

H

2

C

H

C

H

N

H

C

C

N

H

C

C

O

O

O

O

S

O

3

-

S

O

3

-

C

H

2

C

H

2

C

H

C

H

N

H

C

C

N

H

C

C

O

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O

O

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Protein SequencingPreliminary Steps

After breaking disulfide bonds, the chains are separated by disrupting noncovalent interchain interactions with pH extremes, 8 M urea, 6 M guanidium hydrochloride, or high salt

Then the individual protein chains are separated by electrophoresis or chromatography on the basis of size or charge

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Determining AminoAcid Sequence

Once each protein is purified the amino acid sequence is determined by:

1) determining the amino acid composition (how many of each amino acid are in the protein)

2) identifying the amino and carboxyl terminal amino acids

3) cleaving the protein into two or more sets of peptides using specific enzymatic or chemical reagents such as trypsin or cyanogen bromide

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DeterminingProtein Sequence

4) determining the amino acid sequence of each of the peptide fragments

5) determining the entire protein sequence from the sequences of overlapping peptide fragments

6) locating the position of disulfide bridges between cysteines

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Determining Amino Acid Composition

The amino acid composition is determined by:

Hydrolysis with 6N HCl for one to three days

Separating and quantifying individual amino acids by ion exchange HPLC using an amino acid analyzer

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Determining the N-Terminal Amino Acid

The N-terminal amino acid is determined using either chemical reagents or enzymes

Chemical reagents include: Sanger’s reagent dansyl chloride Edman Degradation

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Sanger’s reagent Treat with

dinitrofluorobenzene to form a dinitrophenyl (DNP) derivative of the amino-terminal amino acid

Acid hydrolysis Extract the DNP-derivative

from the acid hydrolysate with organic solvent

Identify the DNP-derivative by chromatography and comparison with standards

N

+

N

+

F

O

O

O

O

N

+

N

+

F

O

O

O

O

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Dansyl chloride (dimethylaminonaphthylenesulfonyl chloride)

Forms a highly fluorescent derivative of the amino-terminal amino acid

Identified by chromatography and fluorescence detection after acid hydrolysis

Highly senstive Best choice when the amount

of protein is limited

N

C

H

3

C

H

3

S

O

O

C

l

N

C

H

3

C

H

3

S

O

O

C

l

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Edman degradation phenylisothiocyanate (phenyl-N=C=S) adds to

N-terminus then acid treatment cleaves the N-terminal amino acid as a PTH derivative

the remaining protein chain is intact and the cycle can be repeated

under ideal conditions the sequence of 30-60 amino acids can be determined

Leucine aminopeptidase enzyme from hog kidney hydrolyzes the N-

terminal peptide bond best with nonpolar amino acids

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Determining theC-Terminal Amino Acid

Hydrazinolysis hydrazine at 100°C cleaves all peptide bonds

forming hydrazides except for the carboxyl terminal

C-terminus reduced with LiAlH4 forms amino alcohol at C-terminus

Carboxypeptidases enzymatic removal of C-terminus Carboxypeptidase A all except proline, arginine and

lysine Carboxypeptidase B only arginine and lysine Carboxypeptidase C any amino acid care required since rate of removal varies with the

type of amino acid

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Peptide Fragments

After determining the amino acid composition and the N & C-terminal amino acids, at least two different sets of protein fragments are needed for sequencing

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Why Use Fragments?

Why is the protein broken into fragments? Why isn’t the protein sequenced directly?

The sequencing methods currently available are only accurate for peptides up to about 20-30 amino acids, 60 under ideal conditions

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Why 2 Sets of Fragments?

Why can't the entire protein amino acid sequence be determined from a single set of peptide fragments obtained by cleavage with a single reagent?

There’s no way to determine how the fragments are connected with just one set

A second or third set of fragments are used to deduce how the fragments are connected by identification and comparison of overlapping sequnces

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Protein Cleavage Reagents

What types of reagents are best suited for preparing these sets of fragments?

Reagents that cleave the protein chain only at a few specific sites forming fragments that are less than 20-30 amino acids in length

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Protein Cleavage Reagents

Chemical or enzymatic reagents can be used to prepare protein fragments

The most commonly used reagents are:

cyanogen bromide various enzymes including

trypsin chymotrypsin clostripain Staphylococcal protease various endopeptidases

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Cyanogen Bromide

At which amino acid in the protein sequence does the reagent, cyanogen bromide, cleave protein chains?

At internal methionines by reaction with the methionine sulfur as illustrated above

N

C

C

H

2

C

O

N

H

H

H

C

H

2

S

C

H

3

N

C

B

r

N

C

C

H

2

C

O

N

H

H

H

C

H

2

S

+

C

H

3

N

C

N

C

C

H

2

C

O

H

H

S

N

C

C

H

2

O

C

H

3

N

C

C

H

2

C

O

N

H

H

H

C

H

2

S

C

H

3

N

C

B

r

N

C

C

H

2

C

O

N

H

H

H

C

H

2

S

+

C

H

3

N

C

N

C

C

H

2

C

O

H

H

S

N

C

C

H

2

O

C

H

3

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Trypsin & Chymotrypsin

Where in the protein sequence do the enzymes, trypsin and chymotrypsin cleave protein chains?

trypsin cleaves at the carboxyl side of amino acids with positively charged side chains such as lysine and arginine

chymotrypsin cleaves at the carboxyl side of amino acids with aromatic side chains such as phenylalanine and tyrosine

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Clostripain

Where in the protein sequence does the enzyme, clostripain, cleave?

prefers positively charged amino acids, arginine even more than lysine

narrower specificity than tryptophan which enzyme is likely to produce

larger fragments?

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Staphyloccal Protease

Where in the protein sequence does the enzyme, Staphylococcal protease cleave?

carboxyl side of acidic amino acids in phosphate buffer

in acetate or bicarbonate buffer it is more specific and cleaves only glutamic acid

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Endopeptidases

The following endopeptidases are less specific than the enzymes metioned above

Pepsin, papain, subtilisin, thermolysin, elastase (papain is the active ingredient in meat

tenderizer, soft contact cleansing solutions, some laundry detergents)

These enzymes are most often used to further reduce the size of large tryptic or chymotryptic fragments

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How are Peptide Fragments Separated?

Usually by column chromatography, often HPLC

Separations are most often based on differences in polarity (reverse phase) or electric charge (ion exchange)

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Edman Degradation

Edman degradation is most often used to sequence the peptides

It removes one amino acid from the N-terminal end of the peptide during each cycle of the procedure

The removal of the N-terminal amino acid is accomplished using the reagent, phenylisothiocyanate

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Edman Degradation

Pheylisothiocyanate attaches to the N-terminal amino acid

The peptide amino nitrogen atom bonds to the PITC carbon

Sulfur then bonds to the peptide carboxyl carbon breaking the peptide bond

This cyclization forms a pheylthiohydantoin derivative which is removed from the peptide chain by treatment with anhydrous acid

Identified by extraction, treatment with aqueous acid and analysis by chromatography

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Edman Degradation

N

C

S

N

H

2

C

C

H

3

C

O

N

H

H

N

H

C

C

H

3

C

O

N

H

H

N

H

C

S

N

H

2

N

N

O

S

C

H

3

H

H

N

C

S

N

H

2

C

C

H

3

C

O

N

H

H

N

H

C

C

H

3

C

O

N

H

H

N

H

C

S

N

H

2

N

N

O

S

C

H

3

H

H

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Disulfide Bridges

The location of disulfide bridges can be determined by diagonal electrophoresis

Fragments with intact disulfide bonds are electrophoresed in one dimension

Treated with fumes of performic acid to cleave disulfide bonds

Then electrophoresed in the second dimension

Fragments that had no disulfide bonds will be on the diagonal

Fragments that had disulfide bonds will migrate off diagonal due to altered mobility

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Mass Spectroscopy

Used for sequencing peptides Peptides are fragmented in the mass

spectrometer The fragments are identified by their

mass/charge ratio Peptide mixtures can be analyzed

using a temperature gradient The temperature gradient causes

variation in signals corresponding to different peptides

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Protein Sequencing by DNA Sequencing

In fact, while you have just learned how to sequece a protein by chemical and enzymatic degradation, protein sequences are now most often determined by translating the corresponding cloned genes

This latter process is usually easier and quicker once the gene corresponding to a given protein has been identified

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Sequence Databases

International databases of protein sequences are maintained

Many of these databases are accessible via the internet

Examples: GenBank Protein Identification Resource (PIR) European Molecular Biology Data

Library (EMBL)

05. Protein Sequencing

Technology

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