Mutational analysis of the three conserved valine residues of insulin and a proposal of "isosteric residue".
To further understand the role of the three conserved Val residues in insulin, B12Val, B18Val, and A3Val, five insulin mutants-[A3Ser]insulin, [B12Thr]insulin, (desB30)[B12Ser]insulin, [B18Thr] insulin, and [B18Leu]insulin--were obtained by means of site-directed mutagenesis and their receptor-binding activities as well as in vivo biological potencies were measured. The two B18 mutants, [B18Thr]insulin and [B18Leu]insulin, both retained relatively high receptor-binding activities (70% and 30% of native porcine insulin, respectively) as well as relatively high in vivo biological potencies. The receptor-binding activities of [B12Thr]insulin and (desB30)[B12Ser]insulin were 5.1% and 0.2%, respectively. However the in vivo biological potency of [B12Thr]insulin was still about 50% of native insulin, whereas that of (desB30)[B12Ser]insulin decreased drastically. The [A3Ser]insulin retained 1.4% of the receptor-binding activity and low in vivo biological potency. These results, together with previous reports showed that when the three conserved Val residues were replaced by residues containing a beta-branched side-chain, such as Thr or Ile, the insulin mutants retained higher biological activities than those mutants replaced by other residues. Here we propose that Val, Thr, and Ile are "isosteric residues' because they all contain a beta-branched side-chain. This proposal may have perhaps general significance in protein design and protein engineering.
Guo
Z Y
ZY
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Tang
Y H
YH
Zhang
Z
Z
Feng
Y M
YM
D000818:Animals; D002942:Circular Dichroism; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000235:genetics*; D007532:Isoleucine / Q000737:chemistry; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013552:Swine; D013912:Threonine / Q000737:chemistry; D014633:Valine / Q000737:chemistry*
D007328:Insulin; D011498:Protein Precursors; D007532:Isoleucine; D013912:Threonine; D014633:Valine