Study On The Influence Of The Hydrophobic Region Of Molecular Chaperon Spy On Its Activity

Molecular chaperones are commonly found in prokaryotic and eukaryotic cells.One of those functions is to prevent the nascent polypeptide chains from misfolding during the assembly process that would result in an inactive structure.Spy is a newly discovered molecular chaperone in recent years,and its chaperone activity has been confirmed.Researchers have found that the concave hydrophobic region of the three-dimensional structure of the Spy molecule plays an important role in the binding of Spy to the substrate protein.In this project,a few hydrophobic amino acids adjacent to the predicted substrate binding sites of Spy were mutated into a hydrophilic amino acid residue,serine,and the protective effect of Spy on the substrate protein Im7 was examined by in vivo activity assay.On the basis of this,the Spy mutants with lower activity were selected,purified,and their chaperone activities were assayed in vitro.The aim is to investigate whether the hydrophobic residues of the molecular chaperone Spy had an effect on the chaperone activity and which of the hydrophobic amino acid residues were the most effective to affect Spy's activity.Spy inactive mutants generated in this project will be a unique tool for future study and finally promote the understanding for the mechanism of action of Spy on substrate proteins.