SMALL ANGLE X-RAY SCATTERING STUDY OF SOLVENT ISOTOPE EFFECT AND PROTEIN STABILI

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们在SSRL提出了小角X射线散射实验(SAXS)来研究H2O-D2O溶剂取代效应对球状蛋白质结构稳定性的影响。蛋白质自然存在于水溶液中。然而，为了避免在有水存在的情况下测量蛋白质的困难，通常使用各种实验技术，如红外光谱、拉曼散射、核磁共振和中子散射，将蛋白质溶解在重水中进行生物物理研究。为了有效，这些研究假设氢同位素替代不会对生物分子结构或动力学产生显著影响，这是基于D2O是造成最轻微扰动的溶剂的想法。然而，实验数据，特别是热力学研究表明，溶剂取代会对蛋白质在溶液中的稳定性产生显著影响。特别是，D2O取代H2O可以稳定蛋白质的热变性和尿素诱导的变性。更重要的是，已发现一些蛋白质在D2O和H2O中似乎采用不同的结构构象，而其他溶液条件相同。小角散射实验是唯一能直接提供蛋白质分子在溶液中整体结构构象的基本信息的技术。它将极大地补充热力学结果，并有助于我们理解氚同位素对蛋白质变性的影响。在这项拟议的项目中，我们将监测两种模型蛋白质--母鸡S鸡蛋溶菌酶和牛血清白蛋白在热变性和尿素诱导变性过程中的结构变化，并研究D2O取代H2O对变性行为的影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose small angle x-ray scattering experiments (SAXS) at SSRL to study the influence of H2O-D2O solvent substitution effect on the structural stability of globular proteins. Proteins naturally exist in aqueous solutions. However, it is common to dissolve proteins in heavy water for biophysical studies using various experimental techniques, e.g., infrared spectroscopy, Raman scattering, nuclear magnetic resonance, and neutron scattering, in order to avoid the difficulties involved in the measurement of proteins with the presence of H2O. To be valid, these studies assume that deuterium isotopic substitution does not influence biomolecular structure or dynamics significantly, based on the idea that D2O is the solvent that causes the mildest possible perturbation. However, experimental data, especially thermodynamic studies, have shown that solvent substitution can cause marked effects on the stability of proteins in solution. In particular, the substitution of D2O for H2O stabilizes the proteins against thermal denaturation and urea-induced denaturation. More significantly, it has been found that some proteins appear to adopt different structural conformations in D2O and H2O, with other solution conditions being identical. The small angle scattering experiment is the only technique that directly provides essential information on the global structural conformation of protein molecules in solution. It will greatly complement the thermodynamic results and contribute significantly to our understanding of deuterium isotopic effects on protein denaturation. For this proposed project, we will monitor the structural change of two model proteins, hen?s egg lysozyme and bovine serum albumin (BSA) dissolved in H2O and D2O, respectively, in the thermal denaturation and urea-induced denaturation processes, and study the influence on the denaturation behavior by the substitution of H2O with D2O.