STRUCTURE--FUNCTION ANALYSIS OF RIBONUCLEASE A

这个研究项目的总体目标是揭示化学物质 蛋白质识别和切割核酸的基础，和 创造出对生物学和医学有用的新型蛋白质。 这项提案的对象是核糖核酸酶A（RNase A），一种小蛋白质 它特异性地催化RNA的P-O 5键的切割， 嘧啶残基。 关于RNase A的经典工作做出了很大贡献 信息酶催化;在化学，折叠， 蛋白质的稳定性;以及分子进化。 这些丰富的 信息，结合产生突变酶的能力， 合成底物类似物，使该系统成为揭示原子 酶催化和蛋白质核酸的决定因素 交互. 本研究项目的具体目标是阐明（1） RNA切割过渡态的结构，（2）切割的机制和 酶的持续合成能力的能量学，（3）静电的重要性 在蛋白质-核酸相互作用中，以及（4）活性位点残基 在催化过程中合作。 此外，杂交蛋白将用于 确定表面环的发散是否允许RNase. A 获得特殊的生物活性。 此外，半合成核糖核酸酶 将被创建，可以遭受特定的网站。 最后，一个最重要的 目的是确定有意义的蛋白质的三维结构， 核酸复合物。 拟议中的实验使用了有机化学的技术， 生物化学，生物物理学和分子生物学，以测试关于 蛋白质结构和蛋白质功能之间的关系（或 功能障碍）。 这些实验的结果将提供新的见解 以及用于生物医学分析的新型蛋白质 和治疗。

The overall objective of this research project is to reveal the chemical basis for the recognition and cleavage of nucleic acids by proteins, and to create novel proteins with properties useful to biology and medicine. The object of this proposal is ribonuclease A (RNase A), a small protein that catalyzes the cleavage of the P-O5 bond of RNA specifically after pyrimidine residues. Classic work on RNase A has contributed much information on enzymatic catalysis; on the chemistry, folding, and stability of proteins; and on molecular evolution. This wealth of information, combined with the ability to produce mutant enzymes and synthesize substrate analogs, make this system ideal for revealing atomic determinants of both enzymatic catalysis and protein - nucleic acid interactions. The specific aims of this research project are to elucidate (1) the structure of the transition state in RNA cleavage, (2) the mechanism and energetics of enzymatic processivity, (3) the importance of electrostatics in protein - nucleic acid interactions, and (4) how active-site residues cooperate during catalysis. In addition, hybrid proteins will be used to determine whether the divergence of surface loops has allowed RNase.A to acquire special biological activities. Also, semisynthetic ribonucleases will be created that can suffer specific site. Finally, a most important aim is to determine three-dimensional structures of meaningful protein - nucleic acid complexes. The proposed experiments use techniques from organic chemistry, biochemistry, biophysics, and molecular biology to test hypotheses on the relationship between protein structure and protein function (or dysfunction). The results of these experiments will provide new insights into this relationship as well as novel proteins for biomedical analyses and therapies.