Site-directed chemical cleavage of the peptide bond in proteins

Anders LeRoy Lund, Purdue University

Abstract

In model dipeptides, the peptide bond on the C-terminal side of an aspartic $\beta$-hydroxamic acid residue (Asp(NHOH)) is cleaved in the presence of a bifunctional buffer; the products are an aspartyl N-hydroxyimide at the C-terminus and a free amino group at the N-terminus. The goal of this study was to determine if the chemistry used to cleave the peptide bond in the dipeptide model could be used to cleave the peptide bond in model proteins. Three different protein models were used. All three models chosen could be esterified at a specific aspartic acid in the protein. To form the $\beta$-hydroxamic acid side chain in the model, the ester of the selectively modified aspartic acid was transamidated with hydroxylamine at pH 9.0. The modified protein was incubated in a bifunctional buffer to determine if cleavage of the peptide bond on the C-terminal side of the aspartic $\beta$-hydroxamic acid residue would occur. The modified proteins were purified with HPLC and were analyzed by various mass spectrometric techniques. This work determined that cleavage of the peptide bond in a protein did not occur. The proteins were modified, and a single hydroxamic acid was incorporated. To understand the effect of the amino acid sequence on peptide bond cleavage, eight peptides were synthesized. The base peptide sequence was E-S-L-A-D(NHOH)-V-Q-A-V. The synthetic peptides varied in size and composition following the aspartic $\beta$-hydroxamic acid residue. Cleavage of the peptide bond was unaffected by the size of the peptide on the N-terminal side of the Asp(NHOH) nor was there any catalytic effect from a proximal hydroxyl or carboxyl group (an adjacent serine or aspartic acid). In contrast, cleavage of the peptide bond was limited by the size of the peptide on the C-terminal side of the Asp(NHOH).

Degree

Ph.D.

Advisors

Loudon, Purdue University.

Subject Area

Biochemistry

COinS