CONFORMATIONAL STABILITY OF GLOBULAR PROTEINS

蛋白质不能由任何氨基酸序列构成。 的 这项技术在健康和其他领域的潜在应用是 几乎是无限的。 因此，我们必须学会预测 氨基酸序列的变化将影响功能、折叠和 蛋白质的稳定性。 我们研究的主要目标是获得 更好地理解有助于蛋白质稳定性的力。 为此，我们计划继续研究单一 在折叠的构象上的氨基酸序列的变化， 展开状态，并对三个相关的折叠热力学 蛋白质：核糖核酸酶Sa、T1和Ba（RNA酶Sa、T1和Ba）。 （RNase Ba 也称为barnase）。它们含有96、104和110个残基; 1、2和 0二硫键;并具有相似的三维结构。 将研究RNase T1和Ba的突变体，以进一步了解 氢键和极性基团埋藏对分子结构的贡献 构象稳定性 RNase Sa的折叠将被研究 详细地，将制备对应于那些已经 特征在于RNA酶T1和Ba。 我们希望能更好地了解 当地环境的影响，即，上下文，在 给定相互作用对稳定性的贡献。 高分辨率 突变蛋白质的结构对于解释这些突变蛋白质是必不可少的。 结果，并将通过X射线晶体学确定。 核磁共振、尺寸排阻色谱和溶剂微扰差 光谱学将用于更好地了解未折叠的 这些蛋白质的状态。 8 M尿素和6 M GdnHCl中的相互作用 导致RNase T1比RNase Ba更不完全地展开 在现有的未折叠状态下， 在生理条件下。 最后，制备了RNase T1的Ser 35- Met突变体， 将分子切割成两个片段：含有α-螺旋的1-35， 和36-104包含大部分B-片层。 这些碎片将被 其特征在于，当在还原条件下组合时， 二硫键不会形成，当二硫键 完整

Proteins can not be constructed with any amino acid sequence. The potential applications of this technology in health and other areas are almost unlimited. Thus, it is essential that we learn to predict how changes in the amino acid sequence will affect the function, folding, and stability of a protein. The primary goal of our research is to gain a better understanding of the forces that contribute to protein stability. To this end, we plan to continue our studies of the effect of single changes in the amino acid sequence on the conformations of the folded and unfolded states, and on the thermodynamics of folding of three related proteins: ribonuclease Sa, T1, and Ba (RNases Sa, T1 and Ba). (RNase Ba is also known as barnase.) They contain 96, 104 & 110 residues; 1, 2 & 0 disulfide bonds; and have similar three-dimensional structures. Mutants of RNases T1 & Ba will be studied to gain further insights into the contribution of hydrogen bonding and polar group burial to the conformational stability. The folding of RNase Sa will be investigated in detail, and mutants will be prepared that correspond to those already characterized in RNases T1 & Ba. We hope to gain a better understanding of the effect of local environment, i.e., the context, on the contribution of a given interaction to the stability. High resolution structures of the mutant proteins are essential for interpreting these results, and will be determined by x-ray crystallography. NMR, size-exclusion chromatography, and solvent perturbation difference spectroscopy will be used to gain a better understanding of the unfolded states of these proteins. The interactions in 8 M urea and 6 M GdnHC1 that cause RNase T1 to unfold less completely than RNase Ba will be identified, and investigated further in the unfolded states that exist under physiological conditions. Finally, a Ser 35-- Met mutants of RNase T1 has been prepared to allow cleavage of the molecule into two fragments: 1-35 containing the a-helix, and 36-104 containing most of the B-sheet. These fragments will be characterized in isolation, when combined under reducing conditions so that disulfide bonds will not form, and when the disulfide bonds are intact.