Peptides - Bonding, Ionization, and Sequencing

Peptide

Two amino acids can combine by removing one water molecule, forming a peptide (one H₂O lost per peptide)

Amino acid residue

The remainder of the amino acid in the peptide, N-terminus (amino group) on the left, C-terminus (carboxyl group) on the right

What catalyzes peptide formation?

Ribosomes, tRNA molecules carry AA to ribosome, which then adds AA one-at-a-time as directed by mRNA template

Number of possible sequences

Total = 20ⁿ, 20 possible AAs at each position (n=number of residues within a peptide)

Ionization of peptides

Groups within a peptide bond cannot ionize

How to find isoelectric points of peptides using pK_a

The group with the lowest pK_a will lose a H⁺ first

Primary structure determination

cDNA sequencing, enzymatic/chemical fragmentation, followed by fragment separation and automated sequencing

How are pure proteins broken into specific sets of peptide fragments?

Enzymes can be used to fragment proteins; trypsin cleaves COOH side of Arg & Lys, chymotrypsin cleaves on COOH side of Phe (F), Tyr (Y), and Try (W)

Cyanogen bromide fragmentation

Cleaves only after Met to produce two peptides at C-terminus

Edman sequencing

R group different for each amino acid, PTH-AA is identified by gas or liquid chromatography, process is repeated over and over for 40-50 cycles, automated sequencing of 5-20 ug peptide/protein

Why do we use chemical sequencing?

Many genes give rise to proteins of different AA sequences, mRNA can be spliced, many proteins are post-translationally modified to form specialized AA, many proteins are post-translationally cleaved