Iavarone Anthony T, Patriksson Alexandra, van der Spoel David, Parks Joel H
Rowland Institute at Harvard, 100 Edwin H. Land Boulevard, Cambridge, Massachusetts 02142, USA.
J Am Chem Soc. 2007 May 30;129(21):6726-35. doi: 10.1021/ja065092s. Epub 2007 May 8.
Measurements of protein unfolding in the absence of solvent, when combined with unfolding studies in solution, offer a unique opportunity to measure the effects of solvent on protein structure and dynamics. The experiments presented here rely on the fluorescence of an attached dye to probe the local conformational dynamics through interactions with a Trp residue and fields originating on charge sites. We present fluorescence measurements of thermal fluctuations accompanying conformational change of a miniprotein, Trp-cage, in solution and in gas phase. Molecular dynamics (MD) simulations are performed as a function of temperature, charge state, and charge location to elucidate the dye-protein conformational dynamics leading to the changes in measured fluorescence. The results indicate that the stability of the unsolvated protein is dominated by hydrogen bonds. Substituting asparagine for aspartic acid at position 9 results in a dramatic alteration of the solution unfolding curve, indicating that the salt bridge involving Lys8, Asp9, and Arg16 (+ - +) is essential for Trp-cage stability in solution. In contrast, this substitution results in minor changes in the unfolding curve of the unsolvated protein, showing that hydrogen bonds are the major contributor to the stability of Trp-cage in gas phase. Consistent with this hypothesis, the decrease in the number of hydrogen bonds with increasing temperature indicated by MD simulations agrees reasonably well with the experimentally derived enthalpies of conformational change. The simulation results display relatively compact conformations compared with NMR structures that are generally consistent with experimental results. The measured unfolding curves of unsolvated Trp-cage ions are invariant with the acetonitrile content of the solution from which they are formed, possibly as a result of conformational relaxation during or after desolvation. This work demonstrates the power of combined solution and gas-phase studies and of single-point mutations to identify specific noncovalent interactions which contribute to protein-fold stability. The combination of experiment and simulation is particularly useful because these approaches yield complementary information which can be used to deduce the details of structural changes of proteins in the gas phase.
在没有溶剂的情况下对蛋白质展开的测量，与在溶液中的展开研究相结合，提供了一个独特的机会来测量溶剂对蛋白质结构和动力学的影响。本文介绍的实验依赖于附着染料的荧光，通过与色氨酸残基以及电荷位点产生的场相互作用来探测局部构象动力学。我们展示了在溶液和气相中伴随微型蛋白质色氨酸笼构象变化的热涨落的荧光测量结果。进行分子动力学（MD）模拟，作为温度、电荷状态和电荷位置的函数，以阐明导致测量荧光变化的染料 - 蛋白质构象动力学。结果表明，未溶剂化蛋白质的稳定性主要由氢键主导。在第9位用天冬酰胺取代天冬氨酸会导致溶液展开曲线发生显著变化，表明涉及赖氨酸8、天冬氨酸9和精氨酸16（+ - +）的盐桥对于色氨酸笼在溶液中的稳定性至关重要。相比之下，这种取代导致未溶剂化蛋白质展开曲线的变化较小，表明氢键是色氨酸笼在气相中稳定性的主要贡献因素。与该假设一致，MD模拟表明随着温度升高氢键数量的减少与实验得出的构象变化焓相当吻合。与通常与实验结果一致的NMR结构相比，模拟结果显示出相对紧凑的构象。未溶剂化色氨酸笼离子的测量展开曲线与形成它们的溶液中的乙腈含量无关，这可能是去溶剂化过程中或之后构象弛豫的结果。这项工作证明了结合溶液和气相研究以及单点突变来识别有助于蛋白质折叠稳定性的特定非共价相互作用的能力。实验和模拟的结合特别有用，因为这些方法产生互补信息，可用于推断气相中蛋白质结构变化的细节。
