DETERMINATION OF PROTEIN STRUCTURE

Contents

Determination of primary structure

Determination of secondary and tertiary protein structures

Determination of primary structure

Determination of aminoacid composition

Degradation of protein or polypeptide into smaller fragments.

Determination of the aminoacid sequence

Determination of amino acid composition in a protein

1. acid or2. alkali treatment 3. enzyme hydrolysis Pronase

Separation and estimation of amino acids: chromatography

Degradation of protein into smaller fragment

liberation of polypeptides

urea or guanidine hydrochlorideperformic acid

Which of the following is a denaturing substance

A GuanosineB GuanidineC Glutamate D Glycine

Breakdown of polypeptides into fragments

trypsin, chymotrypsin, pepsin and elastaseEnzymatic cleavage:

Chemical cleavage: Cyanogen bromide

Number of polypeptides

dansyl chloride. binds N-terminal aminoAcids form dansyl polypeptideon hydrolysis yield N-terminal dansyl amino acid

Determination of amino acid sequence

1-fluoro-2,4-dinitrobenzene (FDNB)

binds with N-terminal amino acids to form Dinitrophenyl (DNP)

derivative of peptide

• This is on hydrolysis yields DNP – amino acids ( N-terminal) and

free amino acids from the rest of the peptide chain.

• Used for identification of N terminal AA

• DNP-AA – identified by Chromatography

Sanger's reagent

Phenyl isothiocyanate

Reacts with N-terminal amino acid

Phenyl thiohydantoin (PTH)-amino acid is liberated

Identified by chromatography

Edmans reagent

Useful in sequencing first 10-30 amino acids

Sequenator

• This is an automatic machine to determine the amino acid

sequence in a polypeptide

• It is based on the principle of Edmans degradation .

• Amino acids are determined sequentially from N-terminal end

• The PTH-amino acid liberated is identified by HPLC.

• Sequenator takes about 2 hours to determine each amino acid.

Reverse sequencing technique • DNA determines the sequence of amino acids in a polypeptide chain

• By analyzing nucleotide sequence of DNA that codes for protein,

translate nucleotide sequence into amino acid sequence.

• Demerits - fails to identify the disulfide bonds

changes that occur in the amino acids after the protein is synthesized.

1. X-ray crystallography

2. Nuclear magnetic resonance (NMR) spectra of proteins

Determination of secondary , tertiary , quarternary protein structures

Electrophoresis

Proteins are separated on the basis OF SIZE BY

A.) SDS-PAGE

B.) HPLC

C) Affinity Chromatography

D) Ion -exchange Chromatography (Pgi June03)

MOLECULAR WEIGHT OF A PROTEIN CAN BE DETERMINED BY

A POLY ACRYLAMIDE GEL ELECTROPHORESIS PAGE

B SODIUM DODECYL SULPHATE PAGE

C ISO ELECTRIC FOCUSSING

D ION EXCHANGE CHROMATOGRAPHY

Protein purification and separation can be done by all except

A. Chromatography

B. Centrifugation

C.Electrophoresis

D. Densitometry

Chromatography Centrifugation

DIFFUSION

IN CHROMATOGRAPHY MASS MOVEMENTS OF THE SUBSTANCE IS DUE TO

A DIFFUSION B ELECTROPHORESISC PAPER CHROMATOGRPHYD OSMOSIS

Haemoglobin electrophoresis

In the case of Sickle Cell hemoglobin ,

replacement of a negatively-charged Glu in the

standard HbA beta-globin by a

neutral Val in HbS 

results in a protein with a slightly reduced negative charge.  

  

Hemoglobin electrophoresis is based on

A molecular weight

B Charge..

C Solubility

D calorimetric properties

gel filtration / diffusion chromatography

BEST METHODProteins Nucleic acids

Cellulose resins have much greater permeability to macromolecular polyelectrolytes

Carboxymethyl cellulose (CM-cellulose) – Cationic exchanger

DEAE cellulose - Anionic exchanger

Cationic exchangers possess negatively charged group,

and these will attract positively charged cations.

“Acidic ion exchange materials”,

because their negative charges result from the ionization

of acidic group.

Anion exchanger

MOLECULAR SEPERATION OF TWO PROTEINS WITH SAME CHARGE CAN BE DONE BY

A ION EXCHANGE CHROMATOGRAPHYB DIALYSISC GEL DIFFUSION CHROMATOGRAPHYD ELECTROPHORESIS

The following separation technique depends on molecular size of the protein

A . Chromatography on carboxymethyl (CM) cellulose column

B. Iso-electric focussing

C. Gel filtration chromatography/ exclusion chromatography

D.Chromatography on di ethyI amino ethyl (DEAE) cellulose column

Hydrophobic chromatography

Affinity chromatography

Gel chromatography

Paper chromatography

In -which type of chromatography, proteins are Bound to another substance

X ray diffraction

The tertiary structure of protein is detected by:

A. X- ray diffraction

B Spectrophotometry

C.Electrophoresis.

D.Chromatography

The substance present in the gall bladder stones or the kidney

A flurosence spectroscopy

B.Electron microscopy

C.Nuclear magnetic resonance

D.X ray diffraction

Mass spectroscopy

Molecular size is assessed by

A SedimentationB Absorption Mass spectroscopyC.LiophilizationD.Salting out

Chromosome walking

Sequence in a Iong Chain of protein is identified by-

A. restriction fragment length polymorphism

B Chromosome walking

C. Leucine Zipper

D. SSOP sequence specific oligonucleotide probes