Primary Structure And Sequencing Of Proteins. (W2, L3)

Front 1

What is usually the first step in determining the amino acid composition of a protein?

Back 1

HYDROLYSE the protein into its amino acid constituents.

Front 2

What are the conditions for total acid hydrolysis of a protein?

Back 2

Amino acid is placed in 6M HCl at 100ºC for 24 hours.

Front 3

What is a key drawback of acid hydrolysis?

Back 3

It only gives the amino acid composition of a protein and not its amino acid sequence i.e. the exact order in which the amino acid residues occur int the protein.

Front 4

From which end is the primary structure of a protein read?

Back 4

The N-terminal to C-terminal. i.e. from the amino "N" end to the carboxyl ,"C", end.

Front 5

Which protein sequencing methods determine the N-terminal amino acid?

Back 5

-Dansyl chloride method.
-Fluorodinitrobenzene method.

Front 6

Which protein sequencing method determines the C-terminal amino acid?

Back 6

-Limited hydrolysis with carboxypeptidase.

Front 7

Which protein sequencing method determines the N-terminal sequence?

Back 7

Edman degradation.

Front 8

Outline the: Dansyl chloride method and fluorodinitrobenzene

Back 8

-The dansyl chloride molecule attacks the NH2 end (N-terminus) of the amino acid and forms a bond.
-Acid hydrolysis is applied.
-the carboxyl end after the first R group is cleaved, resulting in the N-dansyl amino acid and other residues.

-THE SAME PROCESS APPLIES WITH FLUORODINITROBENZENE METHOD.

Front 9

Which chemical specifically cleaves Met (methionine)

Back 9

Cyanogen Bromide (BrCN) cleaves the peptide bond AFTER Met (Methionine).

Front 10

What is an endoprotease?

Back 10

Endopeptidase or endoproteinase are proteolytic peptidases that break peptide bonds of nonterminal amino acids (i.e. within the molecule).

Front 11

Which peptidase cleaves after Phe (Phenylanaline) or Tyr (Tyrosine)?

Back 11

Pepsin.

Front 12

Which protease cleaves after Lys (Lysine) or Arg (Arginine)?

Back 12

Trypsin.

Front 13

Which protease cleaves after Phe (Phenylanaline), Tyr (Tyrosine) or Trp (Tryptophan)?

Back 13

Chymotrypsin.

Front 14

What is the name of the molecule that attacks the N-terminal of a polypeptide in Edman degradation?

Back 14

Phenylisothiocynate.

Front 15

What is the name of the derivative formed after Phenylisothiocynate forms with the N-terminal of the polypeptide?

Back 15

Phenylthiohydantoin amino acid derivative or "PTH"

Front 16

Outline the Edman process.

Back 16

After each degradation step, the PTH-amino acid is identified by HPLC (High Performance Liquid Chromatography).

Front 17

Limitation of the Edman process?

Back 17

Can only be repeated reliably for a maximum of 30 cycles.

Front 18

Outline the strategy for protein sequencing by Edman degradation.

Back 18

1. PURIFY THE PROTEIN- there will likely be many impurities in the sample.

2. REDUCE THE DISULFIDE LINKAGES - e.g. cystine bind to each other making little rings on their own this makes a mess of sequencing.

3. SEPARATE ANY CHAINS - e.g. α-chains and β-chains.

4. CLEAVE THE CHAINS INTO FRAGMENTS - using proteases and chemical cleavage.

5. SEPARATE THE PEPTIDES AND OBTAIN SEQUENCES.

6. IDENTIFY OVERLAPPING PEPTIDE SEQUENCES.

Front 19

How can proteins be modified?

Back 19

amino acids that make up proteins have many different R-groups that react with many other chemicals in the surrounding environment, thus, modifying the protein. This affects the sequencing of proteins by traditional methods.

Front 20

How can proteins with modifications be sequenced reliably?

Back 20

through MASS SPECTROMETRY.

Front 21

How does Mass spectrometry work?

Back 21

Mass spectrometers precisely measure the mass of molecules (molecular mass). They joint two parts:
1. Ion sources.
2. Measuring apparatus.