权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Role of Conserved Proline Residues in Conformation, Function, and Stability of a Protein

保守脯氨酸残基在蛋白质构象、功能和稳定性中的作用

基本信息

批准号：
02680134
负责人：
YUTANI Katsuhide
金额：
$ 1.41万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

To study the role of Pro residues in the conformation, stability, and function of a protein, nine mutant alpha-subunits of tryptophan synthase from Escherichia coli, in which Ala or Gly was substituted for each of six conserved Pro residues(positions 28, 57, 62, 96, 132 and 207)in 10 microorganisms, were constructed.1) The far-UV CD spectra of five mutant alpha-subunits with Ala in place of Pro, the exception being the mutant at position 207(P207A), were identical to the spectrum of the wild-type protein. CD values in the far-UV region were less negative for P207A, indicating that the Pro residue at position 207 plays a role in maintaining the intact structure of the alpha-subunit.2)Scanning calorimetric measurements(DASM4)showed that the stability of each mutant protein relative to that of the wild-type was about the-same for P57A, less for P62A and P132A, and markedly decreased for P96A and P207A ; which are substituted at less mobile positions.3)To understand how the alpha and beta_2 subunits of tryptophan synthase interact to form an alpha_2beta_2 complex and undergo mutual activation, we have investigated isothermal calorimetric titrations (Omega) of wild type beta_2 subunit with wild type alpha subunit and a mutant alpha subunit containing a substitution of Gly for Pro at position 132 show that both the affinity and the exothermic association enthalpy are greatly reduced in the mutant alpha subunit although the stoichiometry of association is unchanged. We conclude that Pro 132 plays a critical role in subunit interaction and in mutual subunit activation.

为了研究Pro残基在蛋白质构象、稳定性和功能中的作用，对大肠杆菌色氨酸合成酶的9个α亚基进行了突变，其中6个保守的Pro残基分别被Ala或Gly取代，（位置28、57、62、96、132和207）。1）用Ala代替Pro的五种突变体α-亚基的远紫外CD光谱，除了207位的突变体（P207 A）之外，与野生型蛋白的谱相同。2）扫描量热法（DASM 4）测定结果表明，突变体蛋白质的稳定性与野生型蛋白质相比，P57 A的稳定性基本相同，P62 A和P132 A的稳定性较低，P96 A、P207 A表达明显降低;其在较不移动的位置被取代。3）为了理解色氨酸合酶的α和β_2亚基如何相互作用以形成α_2 β_2复合物并经历相互激活，我们已经研究了野生型β_3的等温量热滴定（Omega），2亚基与野生型α亚基和在位置132处含有Gly取代Pro的突变α亚基的比较表明，亲和力和放热缔合焓均为在突变体α亚基中大大减少，尽管缔合的化学计量没有改变。我们的结论是Pro 132在亚基相互作用和相互亚基激活中起着至关重要的作用。