CHEMISTRY AND BIOLOGY OF RIBONUCLEASE A

描述(申请人的描述)：本研究的长期目标 这个项目是为了增进我们对蛋白质化学和生物学的理解。这个 拟议的研究重点是核糖核酸酶A(RNaseA)，这是一种可以 有效地催化RNA的P-0键的断裂。经典作品 核糖核酸酶A为蛋白质科学提供了大量信息。在建议的 研究，这些丰富的信息是与现代思想和方法相结合的 实现六个具体目标。 本研究项目的具体目的是：(1)开发Staudinger结扎术 一种制造半合成蛋白质的强有力的新方法；(2)肽键 异构体-确定主链NH对催化的贡献；(3) 非自然模数--确定对构象稳定性的贡献 二硫键等位体，氢键的共价模拟物，复分解作用 交联剂，以及(3)转角模拟；(4)羟基脲抑制剂--创造一种新型的 核糖核酸酶抑制剂；(5)体内功能--确定真正的生理学 胰腺核糖核酸酶和胞浆核糖核酸酶抑制物的作用 蛋白质(与胰腺核糖核酸酶高亲和力结合) 和分析基因敲除小鼠；以及(6)三维结构-在 原子分辨率：信息量最大的蛋白质变体的结构 在这里创造的。 AIMS 1-3使用RNaseA作为模型系统来检验BRED的假设 适用性。特别是，这些目标使用并扩展了新的技术， 将重组DNA技术与合成有机化学相结合创造 含有极具潜在用途的非天然功能的蛋白质。目标 4-6解决有关RNase功能和结构的最有意义的问题 A本身。 拟议中的实验使用了有机化学中的方法和想法， 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，并最终可能导致 用于生物医学分析和治疗的新蛋白质。

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.