INGABIRE MUNYENTWALI ANGELIQUE

BSN 1-8

2018

SEQUENCING OF PROTEINS

Protein sequencing is a technique to determine the amino acid sequence of a protein. It is a method to understand
the structure and function of proteins in living organism. Amino acid sequence determines the eventual three
dimensitional structure of the protein.

What is protein?

All proteins are polymers of amino acid and all these amino acids excepts two have one amino group attached to the
carboxyl group . What is protein Sequencing ? Protein sequencing is the technique to determine the amino acid
sequence of a protein

•Pehr Edman 1947 He found the method to decode the amino acid sequence of a protein using chemicals

•Fredrick Sanger 1955 He was able to present the complete sequence of insulin.

Determination of amino acid composition

Amino acid composition and purity must be known before starting sequencing. The polypeptide chains of multimeric
proteins should be separated and molecular weight of each chain should be measured. The determination of amino
acid is done by hydrolysis, separation & quantitative analysis

Hydrolysis

The peptide is hydrolyzed into its constituent amino acids by heating it in 6N HCL at 110º C for 24 hours.

Separation

Separation of protein is done by chromatography, dialysis etc.

Quantitative Analysis

The amino acid residue of peptides reacts quantitatively with ninhydrin. On heating, an α-amino acids reacts with
two molecules of ninhydrin to yield an intensely coloured product. Purple colour is given in this test by all amino
acids and peptide having a free α-amino group where as proline gives yellow colour.

Mechanism of protein sequencing

•Sanger’s method

•Edman’s method

Sanger's method

 Fredrick sanger was a Nobel Laureate.

 He showed for the first time that amino acids are covalently together by α-amino and α-carboxyl group.
 He suggested stepwise release and identification of amino acid starting from N-terminal.
 He used a reagent fluro-dinitro benzene (FDB)which is commonly called sanger’s reagent.
 The FDB reacts with free NH2 group of N-terminus.
  Upon hydrolysis a yellow coloured dinitrophenol (DNP)-derivative of N- terminal amino acid is produced.
 The DNP amino acid is identified comparing it with a known standard DNP-amino acid by using gel
chromatography
Densyl Chloride
 The Densyl chloride is commonly used because it forms an intensively coloured derivatives.
 That can be detected with high sensitivity that the dinitrophenyl compound .
 It reacts with an uncharged C and N terminal to form a sulfoamide derivatives that is the stable under
condition that by hydrolyzer peptide bonds.
 Although the densyl method for determining the amino terminal residue is sensitive and powerful.

Disadvantage

 It cannot be used repeatedly on the same peptide because the peptide is totally degraded in the acid
hydrolysis step.
Edman degradation
 In 1950 Pehr Edman developed a method of protein sequencing.
 It involves sequential identification of amino acids from N to C termini.
 Phenyl isothyocynate (PTC) reagent is used for the Edman degradation.
 The amino terminal N-terminal residue of a protein can be identifies by reaction the protein with the PTC
that forms a stable covalent link with the free α-amino group prior to hydrolysis with 6M HCl. M E C H A N I S
M 13 SEQUENCING OF PROTEINS
 Phenyl isothiocynate reacts with the uncharged Nterminal amino group of the peptide to form a
phenylthiocarbonyl derivation.
 The labeled N-terminal amino acid can be identified by comparison of its chromatographic properties with
stanrdard fluorodinitro benzene and dansyl chloride.
 Then, under mild acidic the cyclic (PTH) Phenylthio hydantion of the terminal amino acid is librated which
leaves an intact peptide shorted by one amino acid. M E C H A N I S M 14 SE
 The released PTH amino acid is identified by high performance liquid chromatography.
 The sequencing technique has been automated and refined so that upwards of 50 residues from the N-
termines of a protein can be sequenced from picomolequantities of material.
Limitations of Edman reaction

 The Edman degradation proceeds from the Nterminus of the protein it will not work it the N-terminal amino
acid has been chemically modified.
 It also required the use of either guess work or a separate-procedure to determine the position of disulfide
bridges.
Applications of protein sequencing
 Knowledge of the sequence of amino acids in a protein can offer insights into its three dimensional structure
and its function in cellular location.
 Certain amino acid sequences serve as signals that determine the cellular location chemical modification on
an half life of a protein.
 Protein sequences can elucidate the history of life on Earth.
 India
1. Institute of genomics & Integrative Biology (IGIB),New Delhi.
2. The centre for Genomic Application (TCGA) KOLKATA. Abroad 1. Novo Nordisk foundation centre for
protein Research, University of copenhagen. 2. Institute of protein Research of the Ras (IPR RAS)
Pushchino,Moscow , Region.
Protein sequencing is a technique to determine the amino acid sequence of a protein
 it also gives information regarding which conformation the protein adopts
 Discovering the structures and functions of proteins in living organisms is an important tool for
understanding cellular processes
 It allows drugs that target specific metabolic pathways to be invented more easily.
 The first protein sequencing was achieved by Frederic Sanger in 1953. He determined the amino acid
sequence of bovine insulin
  Sanger was awarded the Nobel Prize in 1958.

Problems and solution

You purify a orolem that consist of amino acids .yours goals is to determine which exact sequence of 7 amino acids.you goal is to determine
what the exact sequence of amino acidis

You can explore the protein to two different proteolytic agents ,there by obtaining two sets of fragments

1.a.after exposing the protein to trypsin ,you obtain

Gly-Trp-Arg

Tyr-Lys

asp-ser

b.after exposing the protein to chymotrypsin ,you obtain

Lys-asp-ser

Gly-Trp

Arg-Tyr

Answ; Gly -Trp ↓ Arg -Tyr Lys-Asp-Ser

Gly -Trp-Arg Tyr-Lys Asp-Ser

Therefore the best solution will be Gly-trp-Arg-Tyr-Lys-Asp-Ser

2.If you treat the following peptide either chymotrypsin, which peptide would you except to generate?

Lys-Gly-Phe-Thr-Tyr-Pro-Asn-Trp-Ser-Tyr-Phe

a. Lys-Gly-Phe, Thr-Tyr-Pro-Asn-Trp, Ser-Tyr-Phe

b. Lys-Gly-Phe, Thr-Tyr-Pro-Asn-Trp-Ser-Tyr-Phe
c. Lys-Gly-Phe, Thr-Tyr-Pro-Asn-Trp, Ser-Tyr-Phe
d. Lys-Gly, Phe-Thr-Tyr, Pro-Asn-Trp-Ser-Tyr,Phe
Ans:A

3.You seek to derive amino acid sequencing of a purified peptide. your research has perfomed multiple tests and presents you with the following
data; Ala

Edema degradation (1 cycle): his

Chymotrypsin digestion yields free Tyrp and Ala as well as 1 peptide comprised of Ala,Tyr,and His

Elactase digestion yields a tripeptide and a dipeptide

Your technician makes five predictions about the primary sequencing of the peptide. Which of the following listed below best represent the
unknown peptide

a.NH2-Trp-Tyr-Ala-Ala-His-COOH

b. NH2- Ala-Ala-Trp-Tyr- His-COOH

c. NH2-His-Ala-Tyr-Trp -Ala -COOH

d. NH2-Ala-His-Tyr-Trp -his -COOH

e. NH2- His -Ala -Trp-Tyr -Ala- -COOH

ans:c
4.the standard genetic code is real in codons that are three nucleotides long.how many potential reading frames are there on a single piece of
double stranded DNA ? If instead the genetic code was read in codons that were four nucleotides long ,how many reading frames would be
there be on the same piece of double stranded DNA>

Ans:one strand of DNA has three different overlapping reading frames ,therefore a double -stranded DNA has six reading frames.this can be
seen by examining DNA sequence beginning at the 5’ end of each strand and marking off the triplet codons .this identifies one reading frame on
each strand .now start at the second nucleotide in from the 5’ends and mark off triplet codons ,that is reading frame 2.the third reading frame on
each strand begins at the third nucleotide in from the 5’ends .the fourth reading frame is identical to the first

Using similar logic it follows that if the genetic code were read in codons four nucleotides in length ,then one strand DNA could be read in four
different reading frames and therefore a double stranded piece of DNA would contain eight reading frames(four on each strand).

5.calculate the number of phodphoanhydride bonds that are hydrolyzed during synthesis of 600-amino acid residue protein in E.coli .do not
include the energy required to synthesize the amino acid ,mRNA,tRNA or the Ribososmes

Ans: 2.two phosphoanhydride bonds are hydrolyzed for each amino acid activated byy an aminoacyl-tRNA synthetase.

Amino acid +tRNA+ATP→ Aminoacyl-tRNA +AMP+PPi

PPi+H2O→2 Pi

The rest of the energy to synthesize the protein is provided by hydrolysis of GTP :one high -energy bond is hydrolyzed in the formation of the
70S initiation complex ,an other during insertion of each aminoacyl-tRNA into the A site of ribosome and another at each translocation
step.since the initial methionyl -tRNA is inserted into the Psite,599 new insertions and 599 translocation occur during the synthesis of a 600
-residue protein .finaly ,one phosphoanhydride bond is hydrolyzed during release of the completed polypeptide chain from the ribosome .the
total number of phosphoanyhdride bonds hydrolyzed during synthesis of the protein is

Activation (600 ×2) 1200

Initiation 1

Insertion 599

Translocation 599

Termination 1

Total 2400

6.polypeptide chain elongation on the ribosomes can be broken down into three discrete steps (the microphyle):(1) binding of correct aminoacyl
_tRNA in the ribososme’s a site,(2)Peptide bond formation and (3) translocation.what,specifically ,is it that gets translocated in the third step of
this cycle?

Answer:the answer depends on your frame of reference.for example ,relative to the ribosomes the mRNA and both tRNA get translocated by one
triplet codon.relative to the mRNA it is the ribosome that is shifted by three nucleotide