CONFORMATIONAL STABILITY OF GLOBULAR PROTEINS

现在可以用任何所需的氨基酸构建蛋白质 序列。 该技术在健康领域的潜在应用 而其他领域则几乎没有限制。 因此，它是 我们学会预测氨基酸的变化至关重要 序列将影响功能、折叠和稳定性 蛋白质。 为此，我们计划研究单一的效果 氨基酸序列的变化对构象的影响 折叠和展开状态，构象稳定性，以及 核糖核酸酶 T1 (RNase T1) 折叠的热力学。 我们的主要目标是更好地了解折叠 和展开的构象以及有助于 蛋白质的构象稳定性。 RNase T1 是蛋白质折叠研究的优秀模型。 它是 已知最小的酶，只有 104 个残基，并折叠成 致密的球状构象，其中疏水核心是 夹在 4.5 转 α 螺旋和 4 链反链之间 平行β-片层。 折叠可以用两个二硫键来研究 键完整或断裂，并且可以研究展开的分子 在 25°C 的水中，两个二硫键均断裂。 定点诱变将用于制备设计的突变体 深入了解氢键的贡献，以及 疏水和静电相互作用对构象的影响 RNase T1 的稳定性。 这些物质的构象稳定性 将使用尿素和热解折叠来测量突变体 实验。 将使用以下方法研究折叠的热力学 差示扫描微热量计。 对于大多数 有趣的突变体，折叠的三维结构 蛋白质将使用 X 射线晶体学测定（In 与博士的合作。沃尔弗拉姆·桑格 (Wolfram Saenger) 和乌多·海涅曼 (Udo Heinemann)，以及 将使用多种方法研究未折叠蛋白质的结构 的物理技术。 野生型的未折叠构象 我们将详细研究 RNase T1，因为我们有证据表明 该蛋白质在尿素中解折叠后保留了一些结构。 这些 展开状态将与热展开状态进行比较 以及生理条件下存在的展开状态。 二硫键对未折叠构象的影响 也将被研究。

Proteins can now be constructed with any desired amino acid sequence. The potential applications of this technology in health and other areas are almost unlimited. Consequently, 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. To this end, we plan to study the effect of single changes in the amino acid sequence on the conformations of the folded and unfolded states, in the conformational stability, and on the thermodynamics of folding of ribonuclease T1 (RNase T1). Our primary goal is to gain a better understanding of the folded and unfolded conformations and of the forces which contribute to the conformational stability of proteins. RNase T1 is an excellent model for protein folding studies. It is the smallest enzyme known with just 104 residues, and folds to a compact globular conformation in which the hydrophobic core is sandwiched between a 4.5 turn alpha-helix and a 4-strand anti- parallel Beta-sheet. Folding can be studied with the two disulfide bonds intact or broken, and the unfolded molecule can be studied in water at 25 degrees C both disulfide bonds broken. Site-directed mutagenesis will be used to prepare mutants designed to give insight into the contribution of hydrogen bonding, and hydrophobic and electrostatic interactions to the conformational stability of RNase T1. The conformational stability of these mutant will be measured using urea and thermal unfolding experiments. The thermodynamics of folding will be studied using a differential scanning microcalori-meter. For the most interesting mutants, the three-dimensional structure of the folded protein will be determined using x-ray crystallography (In collaboration with Drs. Wolfram Saenger and Udo Heinemann), and the structure of the unfolded protein will be studied using a variety of physical techniques. The unfolded conformations of wild type RNase T1 will be studied in detail because we have evidence that the protein retains some structure after unfolding in urea. These unfolded states will be compared with the thermally unfolded states and the unfolded states that exist under physiological conditions. The influence of the disulfide bonds on the unfolded conformations will also be studied.