Histidine residues in elongation factor EF-tu from Escherichia coli protected by aminoacyl-tRNA against photo-oxidation.
Complexes of Escherichia coli elongation factor EF-Tu with GTP or GTP and aminoacyl-tRNA were photo-oxidized by irradiation with visible light in the presence of rose bengal dye. EF-Tu was isolated, digested with trypsin, the resulting tryptic peptides were separated by high-performance liquid chromatography (HPLC), and the position of most of the peptides on the chromatogram was determined. Irradiation of complexes resulted in the inactivation of the factor (as tested by its capacity to interact with aminoacyl-tRNA) and was accompanied by the loss of its histidine residues (as revealed by amino acid analysis) and by the decrease in the amount of some tryptic peptides (as detected by HPLC). Aminoacyl-tRNA, bound to EF-Tu during the irradiation, protected the protein from inactivation, from the loss of histidine residues and some of its peptides from photo-oxidative degradation. Comparison of quantities of individual tryptic peptides recovered from the irradiated EF-Tu X GTP X aminoacyl-tRNA complex with those from the irradiated EF-Tu X GTP complex revealed that histidine-containing peptides T12 and T15 as well as methionine-containing peptide T14 were in the ternary complex markedly protected against the photo-oxidative degradation. This finding suggests that their histidines, i.e. His-66 and His-118 respectively and at least one of the methionines (Met-91, 98 or 112) present in peptide T14 are located near to or at the binding site of EF-Tu for aminoacyl-tRNA and could be involved in the interaction between aminoacyl-tRNA and the factor.
D000478:Alkylation; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000378:metabolism; D006639:Histidine / Q000032:analysis* / Q000528:radiation effects; D010084:Oxidation-Reduction; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000378:metabolism / Q000528:radiation effects*; D010777:Photochemistry; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D014357:Trypsin
D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D006639:Histidine; D006160:Guanosine Triphosphate; D014357:Trypsin; D010444:Peptide Elongation Factor Tu