protein sequences 9/21 Flashcards
general method for determining protein sequence with peptides under 50 residues
- amino acid composition
- determine N and C terminal
- label with PITC (edman’s reagent) under alkaline conditions
- treat with 6K HCL
what does sanger’s reagent do
labels N terminal and frees amino group
what are exopeptidases and what are the different types
- enzymatically remove one residue at a time from the end of the chain
- carboxypeptidase and aminopeptidases
what are endopeptidases
cut peptide in middle
what is edman’s reagent
- works for peptides with fewer than 50 residues
- PITC
- alkaline (basic) conditions
- binds to amino terminal
- 6M HCL to break amino acid and form PITC derivative of N terminal amino acid
disulfide bonds
bond between two cysteines
finding amino acid composition for larger peptides and proteins general
- break disulfide bonds with DTT and iodoacetate or performic acid
- cleave the peptide chain with specific endopeptides or cyanogen bromide
- sequences the fragments by edman degradation
- determine the order of the fragments
- determine location of disulfide bonds
trypsin
endopeptidase - K,R - carboxyl side
chymotrypsin
endopeptidase - F,W,Y - carboxyl side - aromatics
Asp-N-protease
endopeptidase - D,E - amino side
cyanogen bromide
endopeptidase - M - carboxyl side
if a larger peptide/protein fragments must be separated by ______ before sequencing
chromatography or electrophoresis
if two sets of fragments are available, the order of the fragments can be determined by
examining the areas of overlap
decoding gene sequences steps
1) partial protein sequence
2) make olionucleotide probe and find mRNA
3) make cDNA and sequence it
partial hydrolysis steps
1) partial hydrolysis until average peptide size is 3 aa
2) determine aa composition of fragments
