ENERGETICS AND MECHANISM OF FOLDING OF RIBONUCLEASE T1

蛋白质现在可以用任何所需的氨基酸序列来构建。这个 这项技术在健康和其他领域的潜在应用包括 几乎是无限的。因此，最重要的是我们要学习 为了预测蛋白质将如何折叠，只需给定氨基酸序列 我们了解到氨基酸序列的变化将如何影响 蛋白质的功能和稳定性。为此，我们建议研究 核糖核酸酶T1(RNaseT1)深度折叠的能量学和机制。 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，折叠成紧凑的 一种球状构象，其中疏水核心夹在 4.5转α-螺旋和4链反平行的β-折叠片层。这个 这里描述的研究的附加特征是：(1)RNaseT1可以是 用0、1或2个完整的二硫键制备，所有这些都可以折叠 以提供具有酶活性的球状构象，以及(2) 构象稳定性可提高4千卡/摩尔以上 NaCl2浓度的变化。 我们计划使用已建立的动力学方法来研究 RNaseT1与0.1和2个完整的二硫键，以及超过一个 构象稳定性的范围。荧光，差分 光谱、酰胺质子交换速率和活度测量将 习惯于跟随折叠。我们希望回答的关键问题是：(1)如何 慢折叠物种的数量和结构性折叠的数量 中间体依赖于完整二硫键的数量？(2)如何 结构中间体的数量和浓度取决于 天然蛋白质的构象稳定性？(3)α-hELIX 或者在折叠的早期阶段首先使用Beta-Sheet形式？ 核糖核酸酶T1的构象稳定性和折叠热力学 0、1或2个完整的二硫键正在用差示方法进行研究 扫描量热法(与Julian Sturtevant博士合作)，以及 通过对尿素和氯化胍的变性曲线进行分析。 这应该会导致更好地理解用于估计的方法 构象稳定性，以及二硫键对构象稳定性的贡献 蛋白质的构象稳定性。通过研究氯化钠和氯化钠的作用 关于其他盐类的展开，我们希望能了解到盐类 使其对核糖核酸酶T1具有显著的稳定性。这可能会给我们带来洞察力 静电相互作用对构象的贡献 球状蛋白的稳定性。

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 of upmost importance that we learn to predict how a protein will fold given just the amino acid sequence and that we learn how changes in the amino acid sequence will effect the function and stability of a protein. To this end, we propose to study the energetics and mechanism of folding of ribonuclease T1 (RNase T1) in depth. 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 antiparallel beta-pleated sheet. The attactive features for the research described here are: (1) RNase T1 can be prepared with 0, 1, or 2 intact disulfide bonds and all of these will fold to give an enzymically active globular conformation, and (2) The conformational stability can be increased by over 4 Kcal/mole through changes in the NaC1 concentration. We plan to use established kinetic methods to investigate the mechanism of folding of RNase T1 with 0. 1, and 2 intact disulfide bonds, and over a range of conformational stabilities. Fluorescence, difference spectroscopy, rates of amide proton exchange, and activity mesurements will be used to follow folding. Key questions we hope to answer are: (1) How does the number of slow folding species and the number of structural intermediates depend on the number of intact disulfide bonds? (2) How does the number and concentration of structural intermediates depend on the conformational stability of the native protein? (3) Does the alpha-h elix or the Beta-sheet form first in the early stages of folding? The conformational stability and thermodynamics of folding of RNase T1 with 0, 1, or 2 intact disulfide bonds is being investigated using differential scanning calorimetry (in collaboration with Dr. Julian Sturtevant), and through an analysis of urea and guanidinium chloride denaturation curves. This should lead to a better understanding of the methods used to estimate conformational stability, and of the contribution of disulfide bonds to the conformational stability of proteins. By studying the effects of NaC1 and other salts on unfolding, we hope to learn the mechanism by which salts cause their remarkable stabilization of RNase T1. This may give us insight into the contribution of electrostatic interactions to the conformational stability of globular proteins.