CHEMISTRY AND BIOLOGY OF RIBONUCLEASE A

描述（申请人描述）：本研究的长期目标 项目是为了提高我们对蛋白质化学和生物学的理解。的 拟议的研究集中在核糖核酸酶A（RNase A），一种小蛋白质， 有效催化RNA的P-0键的裂解。经典作品 RNase A为蛋白质科学提供了大量信息。拟议 研究，这些丰富的信息与现代思想和方法相结合， 实现六个具体目标。 本研究的具体目标是：（1）Staudinger连接-开发 一种强有力的新方法来创造半合成蛋白质;（2）肽键 电子等排-确定主链NH对催化的贡献;（3） 非自然模量-确定对构象稳定性的贡献 二硫键电子等排体，氢键的共价模拟物，复分解 交联，和（3）转向模拟物：（4）羟基脲衍生物-创造一种新型的 核糖核酸酶抑制剂;（5）体内功能--决定真生理活性 胰腺核糖核酸酶和胞浆核糖核酸酶抑制因子的作用 蛋白质（以高亲和力与胰腺核糖核酸酶结合）， 和分析敲除小鼠;和（6）三维结构-确定在 原子分辨率最具信息量的蛋白质变体的结构 在此创建。 目的1 - 3使用RNase A作为模型系统来测试广泛的假设。 适用性特别是，这些目标使用并扩展了新技术， 将联合收割机重组DNA技术与合成有机化学相结合， 含有具有巨大潜在用途的非天然功能的蛋白质。旨在 4 - 6解决了关于RNase功能和结构的最有意义的问题 A自己 所提出的实验使用有机化学的方法和思想， 生物化学、生物物理学、分子生物学和分子遗传学， 对蛋白质化学和生物学的新见解，并可能最终导致 用于生物医学分析和治疗的新型蛋白质。

DESCRIPTION (applicant's description): The long-term objective of this research project is to enhance our understanding of protein chemistry and biology. The proposed research is focused on ribonuclease A (RNase A), a small protein that efficiently catalyzes the cleavage of the P-0 bond of RNA. Classic work on RNase A has contributed much information to protein science. In the proposed research, this wealth of information is combined with modern ideas and methods to achieve six specific aims. The specific aims of this research project are (1) Staudinger ligation-develop a powerful new method for creating semisynthetic proteins; (2) peptide bond isosteres-determine the contribution of a main-chain NH to catalysis; (3) nonnatural modules-determine the contribution to conformational stability of disulfide bond isosteres, a covalent mimic of a hydrogen bond, a metathesis crosslink, and (3) turn mimics; (4) hydroxyurea inhibitor-create a new type of ribonuclease inhibitor; (5) function in vivo-determine the true physiological role of pancreatic ribonuclease and the cytosolic ribonuclease inhibitor protein (which binds to pancreatic ribonuclease with high affinity) by creating and analyzing knockout mice; and (6) three-dimensional structures-determine at atomic resolution the structures of the most informative protein variants created herein. Aims 1 - 3 use RNase A as a model system to test hypotheses of broad applicability. In particular, these aims use and extend new techniques that combine recombinant DNA technology with synthetic organic chemistry to create proteins containing nonnatural functionalities of great potential utility. Aims 4 - 6 address the most meaningful issues on the function and structure of RNase A itself. The proposed experiments use methods and ideas from organic chemistry, biochemistry, biophysics, molecular biology, and molecular genetics to provide new insights into protein chemistry and biology, and could ultimately lead to novel proteins for biomedical analyses and therapies.