Biophysical Characterization Of Macromolecules

在上一次报告所述期间，我们对伽马晶体蛋白的溶液性质和水化作用进行了研究。这些蛋白质代表了脊椎动物眼晶状体细胞核中的主要蛋白质成分。目前，它们的浓度极高，在一些物种中甚至接近紧密堆积的极限。它们的聚集与白内障的形成有关。伽马晶体蛋白在如此高的浓度下保持可溶的生物物理性质还知之甚少。在与格雷姆·威斯托博士的合作下，我们研究了不同物种的晶体蛋白。应用密度对比沉淀速度和密度对比沉淀平衡分析超速离心法，结合基于结构的流体动力学模型和基于结构的水化壳预测，我们能够证明来自最极端环境的伽马晶体蛋白的水化程度异常低，鱼类伽马M晶体蛋白是由蛋白质表面的大量蛋氨酸残基驱动的。相比之下，人的伽马D晶体蛋白表现出正常的蛋白质水化水平。这些结果揭示了水合作用对蛋白质稳定性的贡献与大分子拥挤效应之间的平衡。 在与Mark Mayer博士合作的另一个项目中，我们继续研究不同谷氨酸受体亚型的氨基末端结构域的同源和异源齐聚。这些受体的四级结构控制着它们的离子门控特性。为了表征它们的相互作用，我们应用了沉降速度分析超速离心增强荧光检测，其中谷氨酸受体的高亲和力相互作用作为模型系统。 最后，我们还参与了其他几项研究：与艾米·哈德森博士合作，建立了人类疱疹病毒7型免疫规避分子U21与MHC 1类分子的结合模式，使我们在多信号沉积速度技术的应用方面获得了更多经验；与Alexander Wlowawer博士的实验室合作，我们扩展了SEDPHAT软件的多方法建模能力，使其能够对Fabs结合的三聚体HIV-1 gp41的荧光光谱和恒温滴定热计数据进行全局分析；与Geoffrey Howlett博士合作，我们进一步完善了淀粉样纤维长度分布的进化计算模型；我们与David Marulies博士一起研究了构成多肽负载复合体的分子，该项目有可能提供更多的信息，使我们有可能进化为全球多方法分析的模型应用；与George Patterson博士合作，我们已经着手表征荧光蛋白的自结合特性，这揭示了这些分子在细胞成像中的潜在用途，同时使我们在分析超速离心中的荧光光学检测方面获得了更多经验。

In the last reporting period we have pursued the study of solution properties and hydration of gamma crystallins. These proteins represent the major protein component in the nucleus of vertebrate eye lenses. They are at present extremely high concentrations, in some species even approaching the close packing limit. Their aggregation is associated with the formation of cataract. The biophysical properties by which gamma crystallins can remain soluble at such high concentrations are poorly understood. In collaboration with Dr. Graeme Wistow (NEI) we studied crystallins from different species. Applying density contrast sedimentation velocity and density contrast sedimentation equilibrium analytical ultracentrifugation, in conjunction with structure- based hydrodynamic modeling and structure-based prediction of the hydration shell, we were able to demonstrate unusually low degrees of hydration for gamma crystallins from the most extreme environments, fish gamma M crystallins, driven by a large number of methionine residues at the protein surface. In contrast, human gamma D crystallin exhibits normal levels of protein hydration. These results shed light on the balance between hydration contributions to protein stability, and the effect of macromolecular crowding. In a separate project in collaboration with Dr. Mark Mayer, we have continued to study the homo- and hetero-oligomerization of amino terminal domains of different glutamate receptor isoforms. The quaternary structure of these receptors controls their ion gating properties. To characterize their interactions we have applied sedimentation velocity analytical ultracentrifugation enhanced with fluorescence detection, for which the high-affinity interactions of glutamate receptors serves as a model system. Finally, we have participated in several other studies: In collaboration with Dr. Amy Hudson, we have established the binding mode of human herpes virus 7 immuno-evasive molecule U21 to host MHC class 1 molecules, allowing us to gain more experience in the application of multi-signal sedimentation velocity techniques; in collaboration with the laboratory of Dr. Alexander Wlodawer we have expanded the multi-method modeling capabilities of our SEDPHAT software to permit the global analysis of fluorescence spectroscopy and isothermal titration calorimetry data of Fabs binding trimeric HIV-1 gp41; in collaboration with Dr. Geoffrey Howlett we have further refined our computational model for the evolution of the length distribution of amyloid fibrils; with Dr. David Margulies we have studied molecules constituting the peptide loading complex, a project potentially providing evolving into a model application for global multi-method analysis; and in collaboration with Dr. George Patterson we have embarked on the characterization of self-association of fluorescent proteins, which sheds light on the potential utility of these molecules in cellular imaging, and at the same time allows us to gain more experience with fluorescent optical detection in the analytical ultracentrifuge.