Protein Surface Mutations: Denatured State Stability Effects

[304751]鲍勒将要进行的研究集中在蛋白质的变性状态在调节天然状态和变性状态之间的热力学平衡中的作用。在高溶剂暴露的氨基酸上的突变在iso1 -细胞色素c表面被提出作为探测变性态的能量学的一种手段，与在埋藏的氨基酸突变相比，减少了天然态能量的干扰。在这些位点亲疏水突变将被用来测试异质聚合物理论的预测，这种突变应该稳定变性状态。疏水到亲水的表面突变也将被研究，看看它们是否对变性态能量有相反的影响。组氨酸被认为在变性状态下连接到细胞色素c的血红素。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胍- hc1和热变性技术来表征。可逆二硫突变方法和傅里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。了解使蛋白质的折叠状态相对于未折叠状态稳定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发展蛋白质药物的制药工业具有重要意义。将要开展的工作将通过关注暴露在蛋白质表面的氨基酸的突变，探索有选择地扰乱蛋白质未折叠状态的方法。这项工作将使用从贝克酵母中分离出来的蛋白质iso1 -细胞色素c进行。将使用标准的热学和化学方法来表征展开蛋白质突变形式所需的能量。两种方法，可逆二硫突变和红外光谱，将被用来评估稳定性变化的程度源于蛋白质的未折叠状态。这些实验有可能提供稳定或破坏蛋白质的方法，这些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. ***