Thapar R, Nicholson E M, Rajagopal P, Waygood E B, Scholtz J M, Klevit R E
Biomolecular Structure Center, University of Washington, Seattle 98195-7742, USA.
Biochemistry. 1996 Sep 3;35(35):11268-77. doi: 10.1021/bi960349s.
This paper describes the effect of N-capping substitutions on the structure and stability of histidine-containing protein (HPr). We have used NMR spectroscopy and conformational stability studies to quantify changes in local and global free energy due to mutagenesis at Ser46, the N-cap for helix B in HPr. Previous NMR studies suggested that helix B of Escherichia coli HPr is dynamic as judged by the rate of exchange of amide protons with solvent. Ser46 was chosen because it is the site of regulatory phosphorylation in HPrs from Gram-positive bacteria, and mutation of this residue to an aspartic acid (S46D) in E. coli HPr (Gram-negative) also makes it a poor substrate in the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Therefore, to understand the mechanism of inactivation of E. coli S46D HPr, as well as the effect of mutagenesis on protein stability, we have characterized three mutants of E. coli HPr: Ser46 has been mutated to an Asp, Asn, and Ala in S46D, S46N, and S46A HPrs, respectively. The results indicate that these N-cap replacements have a marked influence on helix B stability. The effect of mutagenesis on local stability is correlated to global unfolding of HPr. The ability of amino acids to stabilize helix B is Asp > Asn > Ser > Ala. In addition, since there are neither large-scale conformational changes nor detectable changes in the active site of S46D HPr, it is proposed that the loss of phosphotransfer activity of S46D HPr is due to unfavorable steric and/or electrostatic interactions of the Asp with enzyme I of the PTS.
本文描述了N端封端取代对含组氨酸蛋白（HPr）结构和稳定性的影响。我们利用核磁共振光谱和构象稳定性研究来量化由于HPr中螺旋B的N端封端位点Ser46发生诱变而导致的局部和全局自由能变化。先前的核磁共振研究表明，根据酰胺质子与溶剂的交换速率判断，大肠杆菌HPr的螺旋B是动态的。选择Ser46是因为它是革兰氏阳性菌HPr中调节性磷酸化的位点，在大肠杆菌HPr（革兰氏阴性菌）中将该残基突变为天冬氨酸（S46D）也使其成为细菌磷酸烯醇丙酮酸：糖磷酸转移酶系统中的不良底物。因此，为了理解大肠杆菌S46D HPr失活的机制以及诱变对蛋白质稳定性的影响，我们对大肠杆菌HPr的三个突变体进行了表征：在S46D、S46N和S46A HPr中，Ser46分别突变为天冬氨酸、天冬酰胺和丙氨酸。结果表明，这些N端封端取代对螺旋B的稳定性有显著影响。诱变对局部稳定性的影响与HPr的全局去折叠相关。氨基酸稳定螺旋B的能力为天冬氨酸>天冬酰胺>丝氨酸>丙氨酸。此外，由于S46D HPr的活性位点既没有大规模的构象变化，也没有可检测到的变化，因此有人提出，S46D HPr磷酸转移活性的丧失是由于天冬氨酸与磷酸转移酶系统的酶I之间不利的空间和/或静电相互作用。
