Perl D, Holtermann G, Schmid F X
Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, D-95440 Bayreuth, Germany.
Biochemistry. 2001 Dec 25;40(51):15501-11. doi: 10.1021/bi011378s.
Residues Arg3 and Leu66 are crucially important for the enhanced stability of the cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus relative to its homologue Bs-CspB from the mesophile Bacillus subtilis. Arg3, which replaces Glu3 of Bs-CspB, accounts for two-thirds of the stability difference and for the entire difference in Coulombic interactions between the two proteins. Leu66, which replaces Glu66 of Bs-CspB, contributes additional hydrophobic interactions. To elucidate the role of these two residues near the chain termini for the rapid folding of the cold shock proteins, we performed an extensive mutational analysis of the folding kinetics to characterize interactions between residues 3, 46, and 66 in the transition state of folding. We employed a pressure-jump apparatus which allows folding to be followed over a broad range of temperatures and urea concentrations in the time range of microseconds to minutes. The N-terminal region folds early, and the interactions that originate from residue 3 are present to a large extent in the transition state already. They include a hydrophobic contribution, a general electrostatic stabilization by the positive charge of Arg3 in Bc-Csp, and a pairwise Coulombic repulsion with Glu46 in the Arg3Glu variant. The C-terminus appears to be largely unfolded in the transition state. The interactions of Leu66, including those with the already structured N-terminal region, are established only after passage through the transition state. The N- and C-termini of the cold shock proteins thus contribute differently to the folding kinetics, although they are very close in space in the folded protein.
相对于嗜温性枯草芽孢杆菌的同源蛋白Bs-CspB，嗜热解淀粉芽孢杆菌的冷休克蛋白Bc-Csp增强的稳定性中，残基Arg3和Leu66至关重要。取代Bs-CspB的Glu3的Arg3，占稳定性差异的三分之二，以及这两种蛋白质之间库仑相互作用的全部差异。取代Bs-CspB的Glu66的Leu66，贡献了额外的疏水相互作用。为了阐明链末端附近的这两个残基对冷休克蛋白快速折叠的作用，我们对折叠动力学进行了广泛的突变分析，以表征折叠过渡态中残基3、46和66之间的相互作用。我们使用了一种压力跳跃装置，它可以在微秒到分钟的时间范围内，在很宽的温度和尿素浓度范围内跟踪折叠过程。N端区域折叠较早，源自残基3的相互作用在很大程度上已经存在于过渡态中。它们包括疏水作用、Bc-Csp中Arg3的正电荷产生的一般静电稳定作用，以及Arg3Glu变体中与Glu46的成对库仑排斥作用。C端在过渡态似乎大部分是未折叠的。Leu66的相互作用，包括与已经形成结构的N端区域的相互作用，只有在通过过渡态后才会建立。因此，冷休克蛋白的N端和C端对折叠动力学的贡献不同，尽管它们在折叠蛋白中的空间位置非常接近。
