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

溶剂同位素效应和蛋白质稳定性的小角X射线散射研究

• 批准号: 8362311
• 负责人: LI FU
• 金额: $ 0.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362311
• 关键词: Adopted; Behavior; Bovine Serum Albumin; Complement; Data; Deuterium; Deuterium Oxide; Funding; Grant; Isotopes; Measurement; Modeling; Molecular Conformation; Monitor; Muramidase; National Center for Research Resources; Neutrons; Nuclear Magnetic Resonance; Principal Investigator; Process; Protein Conformation; Protein Denaturation; Proteins; Radiation; Research; Research Infrastructure; Resources; Roentgen Rays; Solutions; Solvents; Source; Structure; Techniques; Thermodynamics; United States National Institutes of Health; Urea; aqueous; base; cost; egg; globular protein; infrared spectroscopy; research study; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们在SSRL提出小角X射线散射实验（SAXS）来研究H2O-D2O溶剂替代效应对球状蛋白结构稳定性的影响。蛋白质天然存在于水溶液中。然而，通常使用各种实验技术将蛋白质溶解在重水中进行生物物理研究，例如红外光谱、拉曼散射、核磁共振和中子散射，以避免存在存在的蛋白质测量中的困难。 H2O。为了有效，这些研究假设氘同位素取代不会显着影响生物分子结构或动力学，基于 D2O 是引起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，溶剂替代会对溶液中蛋白质的稳定性产生显着影响。特别是，用 D2O 替代 H2O 可稳定蛋白质，防止热变性和尿素诱导的变性。更重要的是，我们发现一些蛋白质在 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直接提供溶液中蛋白质分子整体结构构象基本信息的技术。它将极大地补充热力学结果，并为我们理解氘同位素对蛋白质变性的影响做出重大贡献。对于这个拟议的项目，我们将监测两种模型蛋白质，分别溶解在 H2O 和 D2O 中的鸡蛋溶菌酶和牛血清白蛋白 (BSA) 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。