Collins Marcus D, Quillin Michael L, Hummer Gerhard, Matthews Brian W, Gruner Sol M
Department of Physics, Cornell University, Ithaca, NY 14853, USA.
J Mol Biol. 2007 Mar 30;367(3):752-63. doi: 10.1016/j.jmb.2006.12.021. Epub 2006 Dec 15.
Steric constraints, charged interactions and many other forces important to protein structure and function can be explored by mutagenic experiments. Research of this kind has led to a wealth of knowledge about what stabilizes proteins in their folded states. To gain a more complete picture requires that we perturb these structures in a continuous manner, something mutagenesis cannot achieve. With high pressure crystallographic methods it is now possible to explore the detailed properties of proteins while continuously varying thermodynamic parameters. Here, we detail the structural response of the cavity-containing mutant L99A of T4 lysozyme, as well as its pseudo wild-type (WT*) counterpart, to hydrostatic pressure. Surprisingly, the cavity has almost no effect on the pressure response: virtually the same changes are observed in WT* as in L99A under pressure. The cavity is most rigid, while other regions deform substantially. This implies that while some residues may increase the thermodynamic stability of a protein, they may also be structurally irrelevant. As recently shown, the cavity fills with water at pressures above 100 MPa while retaining its overall size. The resultant picture of the protein is one in which conformationally fluctuating side groups provide a liquid-like environment, but which also contribute to the rigidity of the peptide backbone.
通过诱变实验可以探究空间位阻、电荷相互作用以及许多其他对蛋白质结构和功能至关重要的作用力。这类研究已经产生了大量关于使蛋白质处于折叠状态的稳定因素的知识。要获得更完整的认识，需要我们以连续的方式扰动这些结构，而诱变无法做到这一点。借助高压晶体学方法，现在有可能在连续改变热力学参数的同时探究蛋白质的详细特性。在此，我们详细阐述了含腔突变体T4溶菌酶L99A及其假野生型（WT*）对应物对静水压力的结构响应。令人惊讶的是，该腔对压力响应几乎没有影响：在压力下，WT*中观察到的变化与L99A中几乎相同。该腔最为刚性，而其他区域则有显著变形。这意味着虽然一些残基可能会增加蛋白质的热力学稳定性，但它们在结构上可能也无关紧要。正如最近所表明的，在高于100 MPa的压力下，该腔会充满水，同时保持其整体大小。由此得到的蛋白质图景是，构象波动的侧链基团提供了类似液体的环境，但也有助于肽主链的刚性。
