MAPPING SURFACE CONTACTS IN BIOMACROMOLECULE COMPLEXES

绘制生物大分子复合物中的表面接触图

• 批准号: 6018571
• 负责人: ALAN G MARSHALL
• 金额: $ 18.53万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-11-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018571
• 关键词: carbon; cyclin dependent kinase; deuterium; epitope mapping; infrared spectrometry; interferometry; ion cyclotron resonance spectrometry; macromolecule; protein structure; stable isotope; structural biology; surface property; troponin; tumor suppressor proteins

DESCRIPTION: (Adapted from the applicant's abstract) This project aims to identify and map the surface contacts between two or non-covalently bound proteins by combining three general techniques which the P.I. states that he first devised: (a) isotopic amplifications by expression and purification of proteins doubly-depleted in C-13 and N-15; (b) hydrogen/deuterium exchange of individual proteins and protein complexes in solution followed by rapid quenching, enzymatic cleavage to produce dozens of peptide fragments, and prompt ultra high-resolution mass analysts to determine extend of deuterium incorporation (and thus solvent accessibility to) the backbone of amide protons for each fragment; and (c) various Fourier transform ion cyclotron resonance (FT-ICR) techniques (micro/LC/ESI, on-line desalting, external ion cyclotron accumulation, SWIFT ion selection, digital heterodyne/digital quadrature detection, charge state deconvolution and isotopic deconvolution) needed for unequivocal interpretation of the results. The INK4 family of tumor suppressor proteins (p15,p16,p18,p19) acts in part by forming non-covalent complexes with cyclin-dependent kinases (cdk, 34-37 kDa), and cyclin (34 kDa). Only the smallest of these is accessible by high-resolution NMR. The applicants shall begin by mapping the surface contacts in binary complexes of INK4 protein, cdk, and cyclin and various recombinant variants, and then extend the same approach to epitope mapping of antigen: antibody complexes, to the troponin family of muscle proteins.

描述：(改编自申请者的摘要)本项目旨在 识别并映射两个或非共价结合的表面接触 通过结合三种一般技术，私家侦探声称 他首先设计了：(A)通过表达和 C-13和N-15双缺失蛋白质的纯化； 单个蛋白质和蛋白质复合体的氢/氢交换 在溶液中快速淬火，酶解产生 几十个肽片段，并提示超高分辨率质量 分析员将确定氚的掺入范围(从而确定溶剂 对每个碎片的酰胺质子主干的可及性；和(C) 各种傅里叶变换离子回旋共振(FT-ICR)技术 (Micro/LC/ESI，在线除盐，外部离子回旋积累， 快速离子选择、数字外差/数字正交检测、 电荷态反褶积和同位素反褶积) 对结果的明确解释。 INK4家族抑癌蛋白(p15、p16、p18、p19)参与 部分是通过与细胞周期蛋白依赖性蛋白激酶形成非共价复合物(CDK， 34-37 kDa)和细胞周期蛋白(34 KDa)。只有其中最小的一个可以进入 通过高分辨率核磁共振。申请者应从绘制表面开始 INK4蛋白、CDK和细胞周期蛋白二元复合体中的接触以及各种 重组变异体，然后将相同的方法扩展到表位映射 抗原性：抗体复合体，肌钙蛋白家族。