Kinetics of sulfur labeling of Escherichia coli: Formation of disulfide bonds and free N termini in the cellular proteins

Abstract

1. An estimate has been made of the speed of protein biosynthesis in Escherichia coli using disulfide bond formation as an index of the attainment of final protein conformation. After a period of starvation, the flow of replenished 35SO 2− 4 was followed into cysteine associated with transfer RNA, and then into protein, initially in a reduced, alkylatable form. An average time period of approx. 30 sec is required for the attainment of stable SH:SS ratios in cellular protein, and includes in initial crest of unusually rapid maturation which is attributed to the completion of a backlog of partly completed protein. The average time period for attainment of final protein conformation concluded to be about 20 sec would also appear to encompass the time for polypeptide assembly. The results are consistent with the interpretation that proteins coil in their final conformation while the polypeptide is being elongated. 2. Other sources for the variation of SH:SS ratios of cellular protein have been examined. Towards the late stages of bacterial growth, the ratios rise appreciably, due to contributions of highly reduced proteins from the cell membranes. Although the reductive level of the cellular protein of growing cultures is unaffected by physiological perturbations during and after biosynthesis, the protein synthesized in old cultures can be transiently oxidized with cystine. 3. In a study of sulfur labeling of both the cysteine and methionine of cellular protein, a 30-sec lag period was observed for the initiated N termini of the cell protein to become unmasked and susceptible to alkylation. This is presumed to indicate excision of the N-formyl group of the methionine terminus, approximately within the time required for complete protein synthesis.