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: 2701509
• 负责人: ALAN G MARSHALL
• 金额: $ 18.1万
• 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/2701509
• 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：（改编自申请人的摘要）本项目旨在 识别并绘制两个或非共价结合之间的表面接触 蛋白质结合三个一般技术，其中P.I.的国家 他首先设计了：（a）通过表达进行同位素扩增， 纯化在C-13和N-15中双重耗尽的蛋白质;（B） 单个蛋白质和蛋白质复合物的氢/氘交换 在溶液中，随后快速淬灭，酶促裂解以产生 几十个肽片段，并迅速超高分辨率质量 分析员确定氘掺入的程度（从而确定溶剂 每个片段的酰胺质子骨架的可及性;和（c） 各种傅里叶变换离子回旋共振（FT-ICR）技术 （微/LC/ESI，在线质谱，外部离子回旋累积， SWIFT离子选择，数字外差/数字正交检测， 电荷状态去卷积和同位素去卷积）， 对结果的明确解释。 肿瘤抑制蛋白INK 4家族（p15、p16、p18、p19）在肿瘤发生中起作用。 部分通过与细胞周期蛋白依赖性激酶（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人应开始绘制表面 INK 4蛋白、cdk和细胞周期蛋白的二元复合物中的接触，以及各种 重组变体，然后将相同的方法扩展到表位作图 抗原：抗体复合物，肌钙蛋白家族的肌肉蛋白。