amino acid,proteins and peptide sequencing
TRANSCRIPT
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PRESENTED BY
SUPRIYA KUMARI
MSc Ist Yr
ROLL NO -410LS2055
Department of life science
NATIONAL INSTITUTE OF TECHNOLOGY
GUIDED BY- Dr. SAMIR PATRA SIR
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There are around 20 amino acids and all are amino acids .These are arranged in different sequences in different proteins so they exhibit different functions.
Sequencing of proteins are important tool for understanding cellular processes and allows drugs that target specific metabolic pathways to be invented more easily .It also determines which conformation the protein adopts and extent to which it is complexed with any non peptide molecule.
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FREDER ICK SANG ER first worked out the
sequence of amino acid residues in the polypeptide
chains of the hormone insulin.
The amino acid sequences of thousands of different
proteins from many species have been determined
using principles developed by Sanger .
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1. If the protein contains more than one polypeptide chain,
the chains are separated and purified.
2. Intrachain S-S (disulfide) cross-bridges between cysteine residues in the polypeptide chain are cleaved (If these
disulfides are interchain linkages,then step 2 precedes step 1.)
3. The amino acid composition of each polypeptide chain is
determined.
4. The N-terminal and C-terminal residues are identified.11/26/2010 4
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5 . E ach polypeptide chain is cleaved into smaller fragments,
and the amino acid composition and sequence of
each fragment are determined.
6. Step 5 is repeated, using a different cleavage procedure
to generate a different and therefore overlapping set
of peptide fragments
7. The overall amino acid sequence of the protein is
reconstructed from the sequences in overlapping fragments.
8. The positions of S-S cross-bridges formed between
cysteine residues are located. 11/26/2010 5
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The hormone insulin consists
of two polypeptide chains, A and B, held
together by two disulfide cross-bridges (S-S).
The A chain has 21 amino acid residues and an
intrachain disulfide; the B polypeptide contains
30
amino acids. The sequence shown is forbovine insulin.11/26/2010 7
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A number of methods exist for cleaving
disulfides. An important consideration is to
carry out these cleavages so that the original or
even new S-S links do not form.
Oxidation of a disulfide by performic acid
results in the formation of two equivalents of
cysteic acid. Because these cysteic acid side
chains are ionized SO 3 _ groups, electrostatic
repulsion (as well as altered chemistry) prevents
S-S recombination. 11/26/2010 8
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Oxidative cleavage by reaction with
performic acid.11/26/2010 9
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The peptide is hydrolyzed into its constituent amino
acids by heating it in 6 N HCl at 110°C for 24 hours .
The complex amino acid mixture in the hydrolysate
obtained after digestion of a protein in 6 N HCl can be
separated into the component amino acids by either ion
exchange chromatography.
A comparision of chromatographic pattern with that of standard mixture of amino acid composition
of the peptide is done.
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E nd-group analysis reveals several things.F irst, it identifies the N- and C-terminal
residues in the polypeptide chain. Second,it can be a clue to the number of ends in the protein.
If the protein consists of two or more different polypeptide chains, then more than one end group may be discovered .
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F luorodinitrobenzene ( FD NB) was first used to
identify the N-terminal amino acid as it forms a
stable covalent bond with N-terminal residue of the
amino acid.D absyl chloride is now commonly used because it
forms fluorescent derivatives that can be detected with high sensitivity. It reacts with an uncharged -NH2
group to form a sulfonamide derivative that is stable
under conditions that hydrolyze peptide bonds.
Hydrolysis of our sample dabsyl-peptide in 6 N HCl would yield a dabsyl-amino acid, which could be
identified as dabsyl-alanine by its chromatographic
properties . 11/26/2010 12
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E dman degradation, has become the procedure of
choice. This method is preferable because it allows the
sequential identification of a series of residues
beginning at the N-terminus.In weakly basic solutions, phenylisothiocyanate, or
E dman·s reagent (phenyl-N=C=S), combines with the
free amino terminus of a protein, which can be excised from the end of the polypeptide chain and
recovered as a phenylthiohydantoin (PTH) derivative .
EDMAN DEGRADATION A chemical method devised by Pehr E dman is
usually employed .
This PTH derivative can be identified by
chromato ra hic methods 11/26/2010 15
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N-Terminal analysis using Edman·s reagent, phenylisothiocyanate.Phenylisothiocyanate combines with the N-terminus of a peptide undermildly alkaline conditions to form a phenylthiocarbamoyl substitution. Upon
treatment with TFA (trifluoroacetic acid), this cyclizes to release the N-
terminal amino acid residue as a thiazolinone derivative, but the other
peptide bonds are not hydrolyzed. Organic extraction and treatment
with aqueous acid yield the N-terminal amino acid as a phenylthiohydantoinPTH derivative. 11/26/2010 16
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C-TER MINAL ANALYSIS
F or the identification of the C-terminal residue of
polypeptides, an enzymatic approach is commonly used.
E NZYMATIC ANALYSIS WITH
CA R BOXYP E PTI D AS E S.
Carboxypeptidases are enzymes that cleave amino
acid residues from the C-termini of polypeptides in a
successive fashion.
F our carboxypeptidases are in general use: A, B,
C, and Y.
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carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues
Carboxypeptidase C (from citrus leaves) and carboxypeptidase from yeast act on any C-terminal
residue .
Carboxypeptidase A (from bovine pancreas) works
well in hydrolyzing the Cterminal peptide bond of all residues except proline, arginine, and lysine
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STEPS 5 AND 6. FRAGMENTATION OF THE
POLYPEPTIDE CHAIN
The cleavage methods employed are usually
enzymatic, but proteins can also be fragmented by
specific or nonspecific chemical means (such as partial
acid hydrolysis).
Proteolytic enzymes offer an advantage in that they
may hydrolyze only specific peptide bonds, and this specificity immediately gives information about the
peptide products
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A . TRYPSIN
Trypsin is specific in hydrolyzing only peptide bonds
in which the carbonyl function is contributed by an arginine or a lysine residue
Trypsin cleaves on the C-side of Arg or Lys,
generating a set of peptide fragments having Arg or Lys at their C-termini.
The number of smaller peptides resulting from trypsin action is equal to the total number of Arg
and Lys residues in the protein plus one³the
protein·s C-terminal peptide fragment 11/26/2010 20
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Chymotrypsin shows a strong preference for
hydrolyzing peptide bonds formed by the carboxyl groups of the aromatic amino acids, phenylalanine,
tyrosine,and tryptophan.
B . CHY MOTRYPSIN
Chymotrypsin produces a very different set of
products than trypsin, treatment of separate samples
of a protein with these two enzymes generates fragments whose sequences overlap
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C .CY A NOGEN B RO MIDE
CNBr acts upon methionine residues
The nucleophilic sulfur atom of Met reacts with
CNBr, yielding a sulfonium ion that undergoes a
rapid intramolecular rearrangement to form a cyclic iminolactone.
Water readily hydrolyzes this iminolactone, cleaving
the polypeptide and generating peptide fragments having C-terminal homoserine lactone residues at the
former Met positions .
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STEP 7. RECONSTRUCTION OF THE
OVERALL AMINO ACID SEQUENCE
The sequences obtained for the sets of fragments derived from two or more cleavage procedures are
now compared.
Peptides generated from specific hydrolysis of the polypeptide can be aligned to reveal the overall
amino acid sequence .
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Summary of the sequence analysis of catrocollastatin-C,
a 23.6-k D protein found in the venom of the western D iamondback rattlesnake Crotalus atrox.
Sequences shown are given in the one-letter amino acid code. The overall amino acid sequence (2 16 amino acid residues long) for catrocollastatin-C
as deduced from the overlapping sequences of peptide fragments is shown
on the lines headed CAT-C. The other Lines report the various sequences
used to obtain the overlaps.These sequences were obtained from (a) Nterm.:E dman degradation of the intact proteinin an automated E dman sequenator; (b) M:proteolytic fragments generated by CNBr
cleavage, followed by E dman sequencing of the individual
fragments (numbers denote fragments M 1 through M 5 );
(c) K: proteolytic fragments (K3 through K6) from endopeptidase Lys-C cleavage, followed by E dman sequencing;
(d) E : proteolytic fragments from Staphylococcus
protease ( E 13 through E 15 ) digestion of catrocollastatin sequenced in the
E dman sequenator . 11/26/2010 27
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STEP 8. LOCATION OF DISULFIDE
CROSS-BRIDGES
If the primary structure includes disulfide bonds,their locations are determined in an additional step
after sequencing is completed
A sample of the protein is again cleaved with a reagent such as trypsin, this time without first
breaking the disulfide bonds .
The resulting peptides are separated by electrophoresis and compared with the original set
of peptides generated by trypsin
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F or each disulfide bond, two of the original peptides
will be missing and a new , larger peptide will appear.
The two missing peptides represent the regions of
the intact polypeptide that are linked by the
disulfide bond
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REFERENCES
GA RRE TT AN D G R ISHAM
BIOCH E MIST R Y
BY J ERE MY M B ER G,LUB ER T ST R Y ER
BIOCH E MIST R Y
BY L E HNING ER
WIKIP ED IA
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Exam time has come ,take care.
protect yourself from cold and flu11/26/2010 32