Cotranslational amino-terminal processing of cytosolic proteins. Cell-free expression of site-directed mutants of human hemoglobin.

Abstract

The diversity of protein structure is significantly enhanced by cotranslational and posttranslational modifications. Structural features at the amino terminus are especially important contributors to protein function and stability. Amino-terminal processing of cytosolic proteins includes the cleavage of the initiator methionine and N alpha-acetylation. To better understand the rules that govern these cotranslational events, site-directed mutants of the human beta-globin gene encoding for all 19 amino acid replacements of Val-beta 1 were expressed in a cell-free transcription and translation system. The initiator methionine was 100% cleaved when the side chain of the adjacent residue was relatively small (radius of gyration less than 1.29 A), whereas the initiator methionine was 100% retained when the side chain was relatively large. The extent of N alpha-acetylation ranged from 0 to 100% depending on the amino-terminal sequence. The experimental results in this cell-free system faithfully mimic what occurs in nature as judged by the structures of normal and variant human hemoglobins as well as a broad spectrum of other cytosolic proteins.