Lerch Michael T, López Carlos J, Yang Zhongyu, Kreitman Margaux J, Horwitz Joseph, Hubbell Wayne L
Jules Stein Eye Institute, University of California, Los Angeles, CA 90095; and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095.
Jules Stein Eye Institute, University of California, Los Angeles, CA 90095; and.
Proc Natl Acad Sci U S A. 2015 May 12;112(19):E2437-46. doi: 10.1073/pnas.1506505112. Epub 2015 Apr 27.
Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306-E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron-electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ∼ 3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111-114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2-3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape.
静水压力的施加会使蛋白质构象平衡朝着减小系统体积的方向移动。目前的一种观点认为，体积减小主要是通过空腔水化消除蛋白质内部的空隙或空洞来实现的，不过也有人提出了另一种机制，即由于结构弛豫，空洞被蛋白质侧链填充[López CJ, Yang Z, Altenbach C, Hubbell WL(2013)Proc Natl Acad Sci USA 110(46):E4306 - E4315]。在本研究中，利用定点自旋标记、压力分辨双电子 - 电子共振和高压圆二色光谱，对T4溶菌酶的L99A空腔突变体及其衍生物在高压下消除空腔的机制进行了研究。在L99A突变体中，基态与一种激发态处于平衡状态，该激发态的数量仅占总体的约3%，在这种激发态下，空腔被一个蛋白质侧链填充[Bouvignies等人(2011)Nature 477(7362):111 - 114]。本研究结果表明，在L99A中，天然基态在3 kbar压力下是主要构象，空腔水化显然发生在2 - 3 kbar范围内。然而，在存在降低激发态自由能的额外突变的情况下，压力会强烈地使激发态占主导，从而消除带有天然侧链而非溶剂的空腔。因此，空腔水化和结构弛豫都是压力下消除空腔的机制，哪种机制占主导取决于能量景观的细节。
