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：（根据申请人的摘要进行了改编）该项目的目的是 识别并绘制两个或非共价绑定之间的表面接触 通过结合P.I.的三种通用技术来结合蛋白质指出 他首先设计了：（a）通过表达和 在C-13和N-15中双重脱氧的蛋白质纯化； （b） 单个蛋白质和蛋白质复合物的氢/氘交换 在溶液中，然后快速淬灭，酶促切割以产生 数十个肽片段，并提示超高分辨率质量 分析师确定氘掺入的扩展（因此溶剂 每个片段的酰胺质子骨架的可访问性； （c） 各种傅立叶变换离子回旋共振（FT-ICR）技术 （Micro/LC/ESI，在线脱盐，外部离子回旋子积累， Swift离子选择，数字杂作/数字正交检测， 电荷状态反卷积和同位素解卷积） 对结果的明确解释。 肿瘤抑制蛋白的Ink4家族（p15，p16，p18，p19）起作用 通过与细胞周期蛋白依赖性激酶形成非共价复合物（CDK， 34-37 kDa）和细胞周期蛋白（34 kDa）。只有最小的一个可以访问 由高分辨率NMR。申请人应首先映射表面 Ink4蛋白，CDK和细胞周期蛋白的二元复合物的接触以及各种 重组变体，然后将相同的方法扩展到表位映射 抗原：抗体复合物，肌肉蛋白的肌钙蛋白家族。