protein chemistry-iv anu

39
By Dr Anurag Yadav 1 biochem-fmmc

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By Dr Anurag Yadav

1 biochem-fmmc

• Biomedical importance.

• Steps in determining the amino acid sequence.

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Biomedical importance :

Proteins are physically and functionally complex macro-

molecules, that perform multiple critically important roles.

Proteins are subjected to physical and functional changes that

mirror the life cycle of the organism in which they reside.

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• Typically protein is born at translation,

-matured through post-translational processing events such as

partial proteolysis,

-alternate btwn working and resting state through the

interventions of regulatory factors,

-ages through oxidation, deaminations etc.,

-& dies when it is degraded to its amino acids.

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• Important goal is to :

-identification of the proteins

-& provides both a molecular finger print for its

identification & information that can be used to identify ,

clone gene/ genes that encodes it.

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In order to study the structure and function of protein, the interest of protein molecule must be

isolated in a pure form.

Several methods are available: a. Methods based on solubility properties. b. Methods based on charge properties. c. Methods based on molecular size. d. Methods based on the affinity of protein

for other molecules.

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1st – determine no of peptide chains

2nd – determine AA composition

3rd – identification of N and C terminal AA

4th – sequence edmans degradation(20-30 AA)

5th – very long chain proteins by hydrolysis

6th - position of disulphide bonds

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Determine AA composition

Complete hydrolysis

Chromatographic separation

identification

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Determine number of peptide chains

• Dansyl chloride + N terminal AA of the peptide

• No and nature of dansyl AA determined

• Indicated no of polypeptide chain in the protein

• 2 different peptide chains 2 different dansyl AA

complete hydrolysis by boiling with 6 N HCl at 110 degree for 18-36 hours

under anaerobic conditions

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Identification of N and C terminal AA

• N terminal Dansyl chloride and sanger reagent ( FDNB

– fluorodinitrobenzene )

• C terminal carboxypeptidase A and B

• Continued action of A and B results in removal of AA

from C end

• A – not acts if C end Arginine,proline/lysine

• B – acts only if penultimate residue is proline

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Edman degradation (20-30 AA )

• It is Series of reaction which involves stepwise labeling,

removal & identification of AA from N terminal residue

of an peptide, leaving all other peptide intact is called as

Edman degradation.

• Stepwise removal of AA

• Start from N terminal.

• Determination of AA sequencing of protein.

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• Coupling reaction

• Cleavage reaction

• Conversion reaction

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Coupling reaction

Phenylisothiocyanate ( PITC ) + N terminal amino group of peptide chain

Phenylthiocarbamyl (PTC) derivative of the peptide

PTC washed with organic solvent ( benzene ) to extract excess PITC and side products

Dried under vaccum

inert atmosphere ( to avoid oxidation of sulphur atom of

PITC)

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Cleavage reaction

Dried PTC derivative + anhydrous acid ( trifluroacetic acid )

Cleavage of PTC polypeptide ( peptide bond near PTC )

release original N terminal AA residue (2-anilino-5-thiazoline derivative)

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Conversion reaction

unstable Thiazolinone ( derivative of N terminal AA )

more stable derivative PTH

( isomeric 3 phenyl 2 thiohydantoin )

heating thiazolinone in 1 M HCl at 80 degree for 10 min

PTH - AA is end product of one cycle of edmans degradation

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• Degraded AA identified by reverse HPLC

• A new amino terminal is exposed,

• Procedure is repeated until the entire sequence is determined.

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• All steps + injecting into HPLC column + identification of PTH derivative

Sequencing automated analyzer (sequenator)

that mixes reagent in proper proportion, separate the products, identify them & record results.

run for overnight

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Very long chain proteins

• Very long chain proteins

• small peptides of overlapping sequence

• Purified and Edmans degradation

Hydrolysis by 2 or more different site specific enzymes (partial hydrolysis )

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Protein cleavage and peptide formation

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• Two methods of cleavage

– Chemical method

– Enzymatic method

• ENZYMATIC METHOD: this is done by subjecting the

polypeptide chain to hydrolysis by 2/more different site

specific enzymes.

- Trypsin : hydrolyses peptide bond formed by the carboxyl

group of Lysine/Arginine.

- Chymotrypsin: preferntially acts on the peptide bonds formed

by the carboxyl group of the aa phenylalanine, tyrosine,

tryptophan or leucine.

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CHEMICAL METHOD: cyanogen bromide is used for

cleave peptide bond on the carboxyl end of the

methionine.

• N – bromosuccinimide cleaves tryptophan

• these AA rarely occurs in protein structure

• tends to produce large peptides

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• Native protein cleaved by proteolytic enzymes

• Intact disulphide bridges + small peptides

• Dithiothreitol (DTT) cleaves disulphide bridge

• Cleaved peptides disappears (disulphide bond)

• Reappear as peptides of lower mass

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Ordering an Peptide fragment

• The sequenced peptides may be arranged into complete

sequence of original peptide by generating the overlapping

fragments;

- overlapping fragments are generated.

- overlapping fragments are sequenced.

- overlapping sequence are matched.

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Higher levels of protein structure

• X ray diffraction

• Nuclear magnetic resonance

• Ultraviolet light spectroscopy

• Optical rotatory dispersion

• Circular dichroism

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X ray crystallography

• Crystal of protein is first

produced ( represents 3

dimensional lattice of that

molecule )

• Crystal is mounted inside the

capillary tube with solution

from which crystallized ( to

prevent drying )

• Seal the tube and allow to

pass x ray beams

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• Diffraction data and phase information is collected.

• Using computer electron density map is plotted.

• Using computer graphics known protein sequence is

fitted into electron density map.

• Produce the three dimensional model of a protein.

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NMR spectroscopy

• Measures absorbance of radiofrequency of atomic nuclei

• By knowing the frequency at which particular nucleus absorbs energy

• We can identify functional group available in the molecule

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Mass spectrometry

• MS applicable small nonpolar molecules

• Absolute requirement ions in gas phase

• Fast atom bombardment (FAB) , ESI ,MALDI methods development in ionization technology by introduction of these methods analysis of large charged molecules such as proteins and peptides possible

• Ions corresponding to one peptide is selected in the first analyser colloided with argon gas in a collison cell to generate fragment ions fragment ions thus generated separated according to mass , in a second analyser identified and sequence determined

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• Structure determined only in pure form

• Chromatographic techniques Ion exchange , adsorption ,

partition , size exclusion , affinity , HPLC

• Purity of protein electrophoresis

• Molecular weight mass spectrometry

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Hydrophobicity profile

• Average hydrophobicity per residue against sequence

number

• Averaging is achieved by evaluating and using prediction

algorithms

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