Chalikian T V, Völker J, Anafi D, Breslauer K J
Department of Chemistry Rutgers, The State University of New Jersey, Piscataway, NJ 08855, USA.
J Mol Biol. 1997 Nov 28;274(2):237-52. doi: 10.1006/jmbi.1997.1394.
We report the first protein phase-diagram characterized by a combination of volumetric, calorimetric, and spectroscopic techniques. More specifically, we use ultrasonic velocimetry, densimetry, and differential scanning calorimetry, in conjunction with UV absorbance and CD spectroscopy to detect and to characterize the conformational transitions of alpha-chymotrypsinogen A as a function of both pH and temperature. As judged by the CD spectra, we find that, at room temperature, the protein remains in the native state over the entire pH range investigated (pH 1 to 10). The melting profiles of the native state reveal three distinct pH domains in which protein denaturation produces different final states. Below pH 3.1, we find the heat-induced denatured state of the protein to be molten globule (MG), lacking the native-like tertiary structure, while exhibiting significant secondary structural elements. At neutral and alkaline pH, we find the heat-induced denatured state to be unfolded (U), lacking both tertiary and secondary structures, while being structurally similar to the urea-unfolded state. At intermediate pH values (between pH 3.1 and 7), we find the heat-induced denatured state to exhibit properties characteristic of both the MG and U states. Although at room temperature the protein remains native within the whole pH range studied (pH 1 to 10), our volumetric data reveal that the native state slightly "softens" at low pH, probably, due to pH-induced alterations in electrostatic forces causing the packing of the protein interior at low pH and room temperature to become less "tight". This softening of the protein at low pH is reflected in an 8% increase in the intrinsic compressibility, kM, of the protein "native" state. Our volumetric data also allow us to conclude that the heat-induced MG state retains a liquid-like, water-inaccessible core, with a volume that corresponds to about 40% of the solvent-inaccessible core of the native state. By contrast, our volumetric data are consistent with the U state of the protein being essentially unfolded, with the majority of its constituent atomic groups being solvent exposed and, therefore, strongly hydrated.
我们报告了首个通过体积法、量热法和光谱技术相结合表征的蛋白质相图。更具体地说，我们使用超声测速法、密度测定法和差示扫描量热法，结合紫外吸收光谱和圆二色光谱，来检测和表征α-胰凝乳蛋白酶原A的构象转变与pH值和温度的函数关系。根据圆二色光谱判断，我们发现，在室温下，在所研究的整个pH范围内（pH 1至10），该蛋白质保持天然状态。天然状态的熔解曲线揭示了三个不同的pH区域，其中蛋白质变性产生不同的最终状态。在pH 3.1以下，我们发现蛋白质的热诱导变性状态为熔球态（MG），缺乏类似天然的三级结构，同时呈现出显著的二级结构元素。在中性和碱性pH条件下，我们发现热诱导变性状态为未折叠态（U），既缺乏三级结构也缺乏二级结构，同时在结构上与尿素诱导的未折叠状态相似。在中间pH值（pH 3.1至7之间），我们发现热诱导变性状态表现出熔球态和未折叠态的特征。尽管在室温下该蛋白质在整个研究的pH范围内（pH 1至10）保持天然状态，但我们的体积数据表明，在低pH时天然状态会略有“软化”，这可能是由于pH诱导的静电力变化导致蛋白质内部在低pH和室温下的堆积变得不那么“紧密”。蛋白质在低pH时的这种软化表现为蛋白质“天然”状态的固有压缩性kM增加了8%。我们的体积数据还使我们得出结论，热诱导的熔球态保留了类似液体的、水不可及的核心，其体积约相当于天然状态下溶剂不可及核心的40%。相比之下，我们的体积数据与蛋白质的未折叠态基本一致，其大多数组成原子基团暴露于溶剂中，因此被强烈水合。
