CONFORMATIONAL STABILITY OF GLOBULAR PROTEINS

球状蛋白质的构象稳定性

• 批准号: 6519230
• 负责人: CARLOS N PACE
• 金额: $ 24.93万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519230
• 关键词: X ray crystallography; acidity /alkalinity; calorimetry; chemical stability; circular dichroism; conformation; cytotoxicity; disulfide bond; endoribonucleases; enzyme activity; enzyme structure; fluorescence spectrometry; globular protein; hydrogen bond; ionic bond; ionic strengths; mutant; nuclear magnetic resonance spectroscopy; protein denaturation; protein folding; protein purification; protein sequence; site directed mutagenesis; solubility; thermodynamics

Proteins can now be constructed with any amino acid sequence. The importance of applications of this technology in health and other areas is now clear and limited mostly by our current knowledge and imagination. 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 this project is to gain a better understanding of the forces that contribute to the conformational stability of globular proteins and to use this information to develop methods to increase their stability. This requires a thorough understanding of the structures of the folded and unfolded conformations of proteins and of the effect of changes in structure on the equilibrium between these states. To this end, we will continue to study the effect of small changes in the amino acid sequence on the conformations of the folded and unfolded states, and on the thermodynamics of folding of five microbial ribonucleases: RNase Sa, RNase Sa2, RNase Sa3, RNase T1, and RNase Ba, also known as barnase. Some of the questions we hope to answer are: 1) Can mutations such as Phe yields Tyr that add hydrogen bonds to folded proteins be used to increase their stability? 2) Can mutations such as Gly yields Thr that both reduce the conformational entropy of the unfolded state and bury nonpolar surface area or form hydrogen bonds in the folded state be used to increase protein stability? 3) Can an I to I+4 interaction between glutamine and aspartate side chains on the exposed face of an alpha- helix be used to increase protein stability? 4) Can we double the conformational stability of ab RNase without reducing the enzymic activity? 5) How does the net charge on a protein effect its conformational stability and other physical and chemical properties? 6) What determines the pK values of the ionizable groups in folded and unfolded proteins and how do they effect the conformational stability?

蛋白质现在可以用任何氨基酸序列来构建。这项技术在健康和其他领域的应用的重要性现在已经很清楚，但主要受到我们目前的知识和想象力的限制。 因此，我们必须学会预测氨基酸序列的变化如何影响蛋白质的功能，折叠和稳定性。 该项目的主要目标是更好地了解有助于球状蛋白构象稳定性的力，并利用这些信息开发提高其稳定性的方法。 这就需要彻底理解蛋白质折叠和未折叠构象的结构，以及结构变化对这些状态之间平衡的影响。 为此，我们将继续研究氨基酸序列的微小变化对折叠和未折叠状态构象的影响，以及对五种微生物核糖核酸酶（RNase Sa，RNase Sa 2，RNase Sa 3，RNase T1和RNase Ba，也称为barnase）折叠热力学的影响。我们希望回答的一些问题是：1）Phe产生Tyr的突变是否可以用于增加折叠蛋白质的氢键以增加其稳定性？2)突变，如甘氨酸产生的苏氨酸，既减少了构象熵的展开状态和埋葬非极性表面积或形成氢键的折叠状态，可用于增加蛋白质的稳定性？3)α-螺旋暴露面上谷氨酰胺和天冬氨酸侧链之间的I到I+4相互作用能否用于增加蛋白质稳定性？4)我们能在不降低酶活性的情况下使ab RNase的构象稳定性加倍吗？5)蛋白质上的净电荷如何影响其构象稳定性和其他物理化学性质？6)折叠和未折叠蛋白质中可电离基团的pK值是由什么决定的？它们如何影响构象稳定性？