Protein Surface Mutations: Denatured State Stability Effects

9304751 Bowler将要进行的研究集中在蛋白质的变性状态在调节天然和变性状态之间的热力学平衡中的作用。 突变在高溶剂暴露的氨基酸的表面上的异-1-细胞色素c的提出作为一种手段，探测能量的变性状态，减少干扰，从天然状态的能量相对于在掩埋的氨基酸突变。 在这些位点的亲水性到疏水性突变将用于测试杂聚物理论的预测，即这些突变应该稳定变性状态。 还将研究疏水性至亲水性表面突变，以观察它们是否对变性状态能量具有相反的影响。 组氨酸被认为在变性状态下与细胞色素c的血红素连接。 使用诱变方法来改变组氨酸的序列位置，我们将尝试调节变性状态的大小和形状。 所有突变体的热力学将通过盐酸胍和热变性技术来表征。 可逆二硫键突变方法和傅立叶变换红外光谱将用于直接评估我们的突变对蛋白质变性状态的影响。 了解稳定蛋白质折叠状态相对于未折叠状态的因素是生物化学中的一个中心问题。 能够合理地控制蛋白质的稳定性对于日益开发蛋白质药物的制药工业将是非常重要的。 即将进行的工作将探索选择性地扰乱蛋白质的未折叠状态的方法，通过集中于暴露在蛋白质表面的氨基酸的突变。 这项工作将与蛋白质iso-1-细胞色素c，这将是从面包酵母分离。 将使用标准的热和化学方法来表征展开蛋白质的突变形式所需的能量艾德。 将使用两种方法（可逆二硫键突变和红外光谱法）评价稳定性变化源于蛋白质未折叠状态的程度。 这些实验有可能提供稳定或去稳定蛋白质的方法，该方法不影响蛋白质的折叠结构，因此对生物活性的影响最小。 ***

9304751 Bowler The research to be undertaken focuses on the role of the denatured state of a protein in modulating the thermodynamics equilibrium between the native and denatured state. Mutation at highly solvent-exposed amino acids on the surface of iso-1- cytochrome c is proposed as a means of probing the energetics of the denatured state with reduced interference from native state energetic relative to mutation at buried amino acids. Hydrophilic to hydrophobic mutations at such sites will be used to test the prediction of heteropolymer theory that such mutations should stabilize the denatured state. Hydrophobic to hydrophilic surface mutations will also be investigated to see if they have the opposite effect on the denatured state energy. Histidines are believed to ligate to the heme of cytochrome c in the denatured state. Using mutagenesis methods to vary the sequence position of the histidine we will attempt to modulate the size and shape of the denatured state. The thermodynamics of all mutants will be characterized by both guanidine-HC1 and thermal denaturation techniques. A reversible disulfide mutation method and Fourier transform infrared spectroscopy will be used to directly assess the impact of our mutations on the denatured state of the protein. %%% Understanding the factors that stabilize the folded state of a protein relative to the unfolded state is a central issue in Biochemistry. Being able to rationally control the stability of a protein would be of great importance to the pharmaceutical industry which is increasingly developing protein pharmaceuticals. The work to be undertaken will explore methods of selectively perturbing the unfolded state of a protein, by focussing on mutation of amino acids that are exposed on the surface of a protein. The work will be carried out with the protein iso-1-cytochrome c which will be isolated from Baker's yeast. Standard thermal and chemical methods will be used to characterize the energy requir ed to unfold the mutant forms of the protein. Two methods, reversible disulfide mutation and infrared spectroscopy, will be used to evaluate the degree to which stability changes originate from the unfolded state of the protein. These experiments have the potential to provide methods of stabilizing or destabilizing a protein which do not affect the folded structure of a protein and thus would have minimal impact on biological activity. ***