Scholtz J M
Department of Medical Biochemistry, Texas A&M University, College Station 77843-1114, USA.
Protein Sci. 1995 Jan;4(1):35-43. doi: 10.1002/pro.5560040106.
The conformational stability of the histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been determined using a combination of thermal unfolding and solvent denaturation experiments. The urea-induced denaturation of HPr was monitored spectroscopically at fixed temperatures and thermal unfolding was performed in the presence of fixed concentrations of urea. These data were analyzed in several different ways to afford a measure of the cardinal parameters (delta Hg, Tg, delta Sg, and delta Cp) that describe the thermodynamics of folding for HPr. The method of Pace and Laurents (Pace CN, Laurents DV, 1989, Biochemistry 28:2520-2525) was used to estimate delta Cp as was a global analysis of the thermal- and urea-induced unfolding data. Each method used to analyze the data gives a similar value for delta Cp (1,170 +/- 50 cal mol-1K-1). Despite the high melting temperature for HPr (Tg = 73.5 degrees C), the maximum stability of the protein, which occurs at 26 degrees C, is quite modest (delta Gs = 4.2 kcal mol-1). In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of delta Cp, can be achieved using the method of Chen and Schellman (Chen B, Schellman JA, 1989, Biochemistry 28:685-691). A comparison of the different methods for the analysis of solvent denaturation curves is provided and the effects of urea on the thermal stability of this small globular protein are discussed. The methods presented will be of general utility in the characterization of the stability curve for many small proteins.
利用热变性和溶剂变性实验相结合的方法，测定了枯草芽孢杆菌含组氨酸的磷酸载体蛋白（HPr）的构象稳定性。在固定温度下通过光谱法监测尿素诱导的HPr变性，并在固定浓度的尿素存在下进行热变性实验。这些数据通过几种不同的方式进行分析，以得出描述HPr折叠热力学的关键参数（ΔHg、Tg、ΔSg和ΔCp）。采用Pace和Laurents的方法（Pace CN，Laurents DV，1989，Biochemistry 28：2520 - 2525）来估算ΔCp，并对热诱导和尿素诱导的变性数据进行了全局分析。每种用于分析数据的方法得出的ΔCp值相似（1,170±50 cal mol⁻¹K⁻¹）。尽管HPr的熔点较高（Tg = 73.5℃），但该蛋白在26℃时出现的最大稳定性相当适度（ΔGs = 4.2 kcal mol⁻¹）。在中等浓度尿素存在下，HPr表现出冷变性，因此使用Chen和Schellman的方法（Chen B，Schellman JA，1989，Biochemistry 28：685 - 691）可以获得HPr完整的稳定性曲线，包括对ΔCp的测量。本文提供了分析溶剂变性曲线的不同方法的比较，并讨论了尿素对这种小球蛋白热稳定性的影响。所介绍的方法在表征许多小蛋白的稳定性曲线方面将具有普遍实用性。
