Identification Of Fuction Sites In Trigger Factor

Molecular chaperones play significant roles in newly synthesised polypeptide folding and assembling. They have various functions such as transportion protein,promoting misfolding protein degradation, helping nascent polypeptide chain folding and complex assembling and preventing proteins from aggregating. All the molecular chaperones work together and maintain the cellular protein homeostasis to ensure normal function of the cell.As an ATP-independent molecular chaperone, Trigger factor(TF) is a crucial member of prokaryotic molecular chaperones. By its N-terminal binding to the ribosomal exit tunnel of the 50 S, its C-terminal and ribosome will form a semi-closed space, whose interior surface has many micro-regions with various physicochemical properties, to contain the newly synthesized peptide chains. In this way, the newly synthesized polypeptides can fold correctly in the crowded cytoplasmic environment,and finally form their tertiary structures.In order to elucidate the roles of micro-regions of TF, we mainly generated two kinds of TF mutations in different patches including replacing hydrophilic amino acids with non-functional Alanine and replacing the hydrophobic amino acids with hydrophilic Serine. We cloned these mutant sequence into the vector p QE60, by which TF mutant was co-expressed with adenylate kinase(AK). In this co-expression system, the solubility and activity of AK was used to characterize chaperone activity of TF mutants. In our studies, we found different TF mutations had various effects on the solubility and refolding yield of AK, which indicates the mechanism of TF is complicated and synergistic. These studies will help us to have a much better understanding of the mechanism of TF.