CONFORMATIONAL STABILITY OF GLOBULAR PROTEINS

蛋白质现在可以由任何所需的氨基酸构成。 序列。这项技术在健康领域的潜在应用 而其他地区几乎是无限的。因此，它是 我们必须学会预测氨基酸的变化 序列将影响 蛋白。为此，我们计划研究单一的 氨基酸序列在构象上的变化 折叠和展开状态，构象稳定，以及 核糖核酸酶T1(RNaseT1)折叠热力学 我们的主要目标是更好地了解折叠的 以及展开的构象和作用于 蛋白质的构象稳定性。 RNaseT1是研究蛋白质折叠的一个很好的模型。它是 已知的最小的酶只有104个残基，折叠成一个 疏水核心是紧密的球状构象 夹在4.5圈的阿尔法螺旋和4链的反链之间 平行测试页。折叠可以用两个二硫键来研究 键的完整或断裂，以及未折叠的分子可以被研究 在摄氏25度的水中，两个二硫键都断裂了。 定点突变将被用来准备设计的突变体 以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 核糖核酸酶T1的稳定性。这些化合物的构象稳定性 将使用尿素和热展开来测量突变体 实验。研究折叠的热力学将使用 一种差示扫描微卡计。最多的 有趣的突变体，折叠的三维结构 将使用x射线结晶学(In)测定蛋白质 与Wolfram Saenger博士和Udo Heinemann博士的合作)，以及 未折叠的蛋白质的结构将用一种 体能技术。野生型的展开构象 我们将详细研究RNaseT1，因为我们有证据表明 该蛋白质在尿素中展开后仍保持一定的结构。这些 展开态将与热展开态进行比较 以及在生理条件下存在的展开状态。 二硫键对未折叠构象的影响 也将进行研究。

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.