PROTEIN STUDIES WITH UNNATURAL AMINO ACIDS

使用非天然氨基酸进行蛋白质研究

• 批准号: 2186773
• 负责人: PETER G SCHULTZ
• 金额: $ 16.15万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186773
• 关键词: X ray crystallography; aminoacid analog; biophysics; chemical kinetics; chemical models; chemical stability; chemical substitution; circular dichroism; computer simulation; conformation; crystallization; enzyme mechanism; fluorescence spectrometry; hydrogen bond; hydropathy; intermolecular interaction; lysozyme; molecular dynamics; mutant; nuclear magnetic resonance spectroscopy; nuclease; protein engineering; protein folding; protein sequence; protein structure function; site directed mutagenesis; synthetic protein; thermodynamics

Proteins are at the crossroads of all biological processes including molecular recognition, catalysis, signal transduction, information storage and mobility. Yet, we have little understanding of the factors that control the structure, stability, folding and function of proteins. X-ray crystallography and site-directed mutagenesis have proven to be extremely powerful tools for analyzing protein structure/function. However the restrictions imposed on the latter technique by the natural twenty amino acids limit its usefulness. Recently, we have succeeded in developing a general biosynthetic method which, for the first time, makes it possible to site-specifically incorporate unnatural amino acids with novel steric and electronic properties into proteins. Our ability to insert amino acids with a wide range of side-chain and backbone structures into proteins allows us for the first time to carry out precise "physical organic" chemistry on this important class of molecules. Our objectives during the next three years will focus (a) on testing the scope of this new methodology with regard to the kind of amino acid structures that can be incorporated into proteins and (b) using this methodology to analyze protein structure and function. We will (i) undertake a systematic study of those factors that determine protein stability in the model system, T4 lysozyme, including the effects of hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals interactions and entropy on protein thermal stability and (ii) explore the catalytic mechanism of staphylococcal nuclease in an effort to understand in greater detail how this enzyme achieves its remarkable catalytic advantage. In addition, we will develop new tools for analyzing protein structures (folded and unfolded), protein-protein interactions and conformational changes including the site specific incorporation of fluorescent, spin labelled and isotopically labelled amino acids for biophysical studies of protein structure.

蛋白质处于所有生物过程的十字路口，包括 分子识别、催化、信号转导、信息 存储和移动性。 然而，我们对其中的因素知之甚少 控制蛋白质的结构、稳定性、折叠和功能。 X 射线晶体学和定点突变已被证明 用于分析蛋白质结构/功能的极其强大的工具。 然而，自然条件对后一种技术的限制 二十种氨基酸限制了它的用途。 最近，我们成功地 开发一种通用的生物合成方法，首次使 可以位点特异性地掺入非天然氨基酸 蛋白质的新颖空间和电子特性。 我们的能力 插入具有广泛侧链和主链的氨基酸 将结构转化为蛋白质使我们第一次能够进行 精确的“物理有机”化学对这一重要类别 分子。 我们未来三年的目标将集中于 (a) 测试这种新方法的范围 可以整合到蛋白质中的氨基酸结构和（b） 使用这种方法来分析蛋白质的结构和功能。 我们 将 (i) 对那些决定因素进行系统研究 模型系统 T4 溶菌酶中的蛋白质稳定性，包括效果 氢键、静电相互作用、疏水性和范德 瓦尔斯相互作用和熵对蛋白质热稳定性的影响以及 (ii) 努力探索葡萄球菌核酸酶的催化​​机制 更详细地了解这种酶如何实现其卓越的功效 催化优势。 此外，我们还将开发新工具 分析蛋白质结构（折叠和未折叠）、蛋白质-蛋白质 相互作用和构象变化，包括位点特异性 掺入荧光、自旋标记和同位素标记 用于蛋白质结构生物物理学研究的氨基酸。