Mutagenesis of the three conserved valine residues: consequence on the foldability of insulin.
Natural polypeptide chain usually can spontaneously fold into tightly compact native structure. This capability is the so-called foldability. However, how the foldability is encoded in the polypeptide chain is still poorly understood. The structure of insulin has been well solved and extensively investigated. Therefore, insulin provides a good model for investigating the role of individual residue to the sequence foldability. In insulins from different species there are three highly conserved Val residues (A3Val, B12Val, and B18Val), but their contribution to the insulin foldability is still unknown. Here, a single-chain insulin (PIP) was used to investigate the contribution of the three conserved valine residues to the foldability. Five PIP mutants, [A3S]PIP, [A3T]PIP, [B12A]PIP, [B18T]PIP, and [B18L]PIP, were used in the studies, and their structural changes, secretion efficiency, structural stability, disulfide stability, and in vitro refolding efficiency were analyzed. The effects of the mutations on the PIP foldability are multifold: as a whole, mutation of A3Val has only moderate effect; while mutation of B12Val has significant detriment; hydrophobic replacement of B18Val is more tolerant than hydrophilic substitution as foldability is concerned. Therefore, the three highly conserved valine residues have different contributions to the insulin foldability, and their contribution might be ranked as B12Val>B18Val>A3Val.
Guo
Zhan-Yun
ZY
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Wang
Shuai
S
Tang
Yue-Hua
YH
Feng
You-Min
YM
D002942:Circular Dichroism; D017124:Conserved Sequence; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014633:Valine / Q000737:chemistry*
D004220:Disulfides; D007328:Insulin; D014633:Valine