CONFORMATIONAL STABILITY OF GLOBULAR PROTEINS

蛋白质现在可以用任何所需的氨基酸来构建 顺序 这项技术在健康领域的潜在应用 其他领域几乎是无限的。 根据该结果 我们必须学会预测氨基酸的变化 序列将影响功能，折叠，和稳定性， 蛋白 为此，我们计划研究单一 氨基酸序列的变化对构象的影响 折叠和未折叠状态，处于构象稳定性，和 核糖核酸酶T1（RNase T1）的折叠热力学。 我们的主要目标是更好地了解折叠的 和未折叠的构象以及有助于 蛋白质构象的稳定性。 RNase T1是研究蛋白质折叠的理想模型。 是 已知的最小的酶只有104个残基， 紧凑的球状构象，其中疏水核心是 夹在4.5圈α-螺旋和4链反- 平行β片层 折叠可以用两个二硫化物来研究， 键完整或断裂，可以研究未折叠的分子 在25摄氏度的水中，两个二硫键都断裂。 定点诱变将用于制备设计的突变体。 以深入了解氢键的贡献， 疏水和静电相互作用对构象 RNase T1的稳定性 这些分子的构象稳定性 突变体将使用尿素和热解折叠来测量 实验 折叠的热力学将使用 差示扫描微量热计。 在大多 有趣的突变体，折叠的三维结构 将使用X射线晶体学（In 与Wolfram Saenger和Udo Heinemann博士合作）， 未折叠蛋白质的结构将使用多种方法进行研究。 物理技术。 野生型的未折叠构象 RNase T1将被详细研究，因为我们有证据表明， 该蛋白质在尿素中展开后保留一些结构。 这些 将未折叠状态与热未折叠状态进行比较 以及在生理条件下存在的未折叠状态。 二硫键对非折叠构象的影响 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.