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

为了研究Pro残基在蛋白质构象、稳定性和功能中的作用，构建了9个突变的大肠杆菌色氨酸合成酶α亚基，其中Ala或Gly分别替换了10个微生物中6个保守的Pro残基(第28、57、62、96、132和207位)中的每一个。1)5个突变的α亚基的远紫外圆二色谱与野生型蛋白的光谱相同，其中第207位突变的P207A除外。远紫外区的Cd值对P207A的负值较小，表明207位的Pro残基对维持α-亚基的完整结构起到了一定的作用。2)扫描量热法(DASM4)结果表明，每个突变蛋白相对于野生型的稳定性-P57A相同，P62A和P132A较小，而P96A和P207A则明显降低；3)为了了解色氨酸合成酶的α和β_2亚基是如何相互作用形成α_2beta_2复合体并相互激活的，我们研究了野生型β_2亚基与野生型α亚基和突变的α亚基在132位用甘氨酸取代Pro的等温量热滴定(Omega)，结果表明，尽管结合的化学计量学不变，但突变的α亚基的亲和力和放热结合热都大大降低。我们认为，Pro 132在亚基相互作用和相互亚基激活中起着关键作用。

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.