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THREE-DIMENSIONAL ELECTROPHORETIC NMR OF MIXED PROTEINS

混合蛋白质的三维电泳核磁共振

基本信息

批准号：
6581142
负责人：
QIUHONG HE
金额：
$ 4.91万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-09-30 至 2002-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (Adapted from the applicant's abstract): The objective of this proposal is to develop 3D electrophoretic NMR spectroscopy and to identify protein folding intermediates with the technique. At present, NMR methods are capable of characterizing structures of pure proteins, strongly bound protein complexes, and the limited protein mixtures that can be resolved by chemical shifts. However, these methods cannot resolve resonances of multiple protein mixtures with overlapping NMR signals. The proposed novel methods will extend NMR technology for structural determination to meet these challenges. Electrophoretic mobility-ordered, homonuclear-ordered coherence correlation spectroscopy will be developed. By applying a pulsed electric field and magnetic field gradients, the investigators will introduce a dimension of electrophoretic mobility to the homonuclear-correlated NMR spectroscopy. These added dimensions will allow two-dimensional (2D) NMR spectra of different proteins to be sorted according to their electrophoretic mobilities. The plans are to accomplish the following specific aims: 1) develop 3D electrophoretic mobility-ordered correlation spectroscopy (COSY), double-quantum filtered COSY (DQF-COSY), total correlation spectroscopy (TOCSY), and nuclear overhauser enhancement spectroscopy (NOESY) techniques, demonstrating these methods with well-characterized zinc finger peptides, isozyme, and ubiquitin; 2) identify protein folding intermediates and simultaneously observe NMR spectra of co-existing conformations using alpha-lactalbumin (aLA) molten globule as an example. If proven, the development and application of the proposed novel NMR methods may have significant impact to many fields including: protein-protein or protein-DNA interactions, protein folding, enzyme kinetics, and structure-guided protein drug design.

描述（改编自申请人的摘要）：本申请的目的建议是开发3D电泳NMR光谱，并确定蛋白质折叠中间体的技术。目前，NMR方法能够表征纯蛋白质的结构，蛋白质复合物，以及有限的蛋白质混合物，可以解决化学位移然而，这些方法不能解决具有重叠NMR信号的多种蛋白质混合物。拟议中的小说方法将扩展NMR技术用于结构测定，以满足这些挑战。电泳迁移率有序，同源核有序将开发相干相关光谱学。通过施加脉冲电场和磁场梯度，研究人员将引入电泳迁移率的维度，同核相关NMR光谱。这些增加的维度将允许待分选的不同蛋白质的二维（2D）NMR光谱根据它们的电泳迁移率。这些计划是为了实现具体目标如下：1）开发3D电泳迁移率有序相关光谱（COSY），双量子滤波COSY（DQF-COSY），总相关光谱（TOCSY）和核奥弗豪泽增强光谱（NOESY）技术，证明这些方法与充分表征的锌指肽、同工酶和泛素; 2）鉴定蛋白质折叠中间体，同时观察NMR光谱，使用α-乳白蛋白（aLA）熔融球作为共存构象 example. 如果得到证实，开发和应用所提出的新的 NMR方法可能对许多领域产生重大影响，包括：蛋白质-蛋白质或蛋白质-DNA相互作用，蛋白质折叠，酶动力学和结构指导的蛋白质药物设计。