Ercole Carmine, Avitabile Francesca, Del Vecchio Pompea, Crescenzi Orlando, Tancredi Teodorico, Picone Delia
Dipartimento di Chimica, Università di Napoli Federico II, Italy.
Eur J Biochem. 2003 Dec;270(23):4729-35. doi: 10.1046/j.1432-1033.2003.03872.x.
Bovine seminal ribonuclease (BS-RNase) is the only known dimeric enzyme characterized by an equilibrium between two different 3D structures: MxM, with exchange (or swapping) of the N-terminal 1-20 residues, and M=M, without exchange. As a consequence, the hinge region 16-22 has a different tertiary structure in the two forms. In the native protein, the equilibrium ratio between MxM and M=M is about 7 : 3. Kinetic analysis of the swapping process for a recombinant sample shows that it folds mainly in the M=M form, then undergoes interconversion into the MxM form, reaching the same 7 : 3 equilibrium ratio. To investigate the role of the regions that are most affected structurally by the swapping, we expressed variant proteins by replacing two crucial residues with the corresponding ones from RNase A: Pro19, within the hinge peptide, and Leu28, located at the interface between subunits. We compared the structural properties of the monomeric forms of P19A-BS-RNase, L28Q-BS-RNase and P19A/L28Q-BS-RNase variants with those of the parent protein, and investigated the exchange kinetics of the corresponding dimers. The P19A mutation slightly increases the thermal stability of the monomer, but it does not alter the swapping tendency of the dimer. In contrast, the L28Q mutation significantly affects both the dimerization and swapping processes but not the thermal stability of the monomer. Overall, these results suggest that the structural determinants that control the exchange of N-terminal arms in BS-RNase may not be located within the hinge peptide, and point to a crucial role of the interface residues.
牛精核糖核酸酶（BS-RNase）是唯一已知的二聚体酶，其特征在于两种不同三维结构之间的平衡：MxM，N端1-20个残基发生交换（或互换）；M=M，无交换。因此，铰链区16-22在两种形式中具有不同的三级结构。在天然蛋白质中，MxM和M=M之间的平衡比例约为7:3。对重组样品的互换过程进行动力学分析表明，它主要以M=M形式折叠，然后转化为MxM形式，达到相同的7:3平衡比例。为了研究在结构上受互换影响最大的区域的作用，我们通过用核糖核酸酶A的相应残基替换两个关键残基来表达变体蛋白：铰链肽内的Pro19和位于亚基之间界面处的Leu28。我们比较了P19A-BS-RNase、L28Q-BS-RNase和P19A/L28Q-BS-RNase变体单体形式与亲本蛋白的结构特性，并研究了相应二聚体的交换动力学。P19A突变略微增加了单体的热稳定性，但没有改变二聚体的互换趋势。相比之下，L28Q突变显著影响二聚化和互换过程，但不影响单体的热稳定性。总体而言，这些结果表明，控制BS-RNase中N端臂交换的结构决定因素可能不在铰链肽内，并指出了界面残基的关键作用。
