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Allosteric Transition in Lactose Repressor Protein

乳糖阻遏蛋白的变构转变

基本信息

批准号：
7928481
负责人：
KATHLEEN S MATTHEWS
金额：
$ 4.61万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2009-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7928481
关键词：
Adopted Amino Acid Substitution Amino Acids Behavior Binding Binding Sites Biochemical Biological C-terminal Characteristics Chemicals Communication Complex Coupled Crystallization DNA DNA Binding Data Employee Strikes Environment Event Exhibits Family Generic Drugs Genes Genetic Genetic Transcription Goals Hand Homologous Gene Individual Lactose Libraries Ligand Binding Ligands Link Modeling Modification Molecular Conformation Mutation NMR Spectroscopy Organism Pathway interactions Pattern Physiological Property Protein Family Proteins Recording of previous events Regulation Repressor Proteins Research Personnel Resolution Role Side Signal Transduction Site Site-Directed Mutagenesis Structure System Testing TimeLine Transcription Repressor/Corepressor Variant base biological systems comparative conformational conversion design dimer insight member molecular dynamics molecular size monomer mutant novel programs protein aminoacid sequence protein folding protein function protein structure research study response tool

项目摘要

DESCRIPTION (provided by applicant): Biological systems universally employ cascades of binding/catalytic events to transmit information regarding environmental and physiological states. This communication network requires that protein function be altered by allosteric mechanisms. The dearth of atomic level insight into such intramolecular signal transduction motivates our continued studies of the "switching" pathway in the well-studied lactose repressor protein (LacI). In LacI function, ligand binding information must flow through the protein structure between the widely separated inducer and DNA binding sites. Our ultimate goal is an atomic-level view and sequential timeline of the conformational changes that result in release of operator DNA and consequent transcription of downstream genes. We recently used targeted molecular dynamics simulation (TMD) to predict an atomic-level allosteric pathway that is concordant with the large library of experimental data on LacI. Coupled with available phenotypic information, this model provides a unique framework for designing novel experiments to examine and redesign the conformational behavior of LacI and to evaluate TMD as a general tool for deciphering allosteric pathways. Experiments are proposed to examine specific features of LacI: (1) the potential for a single (or only a few) amino acid(s) to trigger allosteric response to inducer; (2) the role of the core N-subdomain interface in adopting the alternate conformations; (3) the regulation of structural shifts by amino acids in the 3-stranded core pivot that links the N- and C-subdomains of the LacI core; and (4) the influence of symmetry on the allosteric response. Site-specific mutagenesis will be used in concert with detailed biophysical analysis (and, where possible, structural data) to assess the role of specific amino acids in the allosteric pathway and the influence of asymmetric amino acid changes within a dimer. LacI provides a unique system with the requisite background of information and tools to test rigorously the predictions of TMD calculations. With established reliability, TMD can be used to more fully exploit the increasing number of end-point structures available for allosteric proteins. Finally, information on allosteric changes in LacI can be integrated with the recent successful design of novel ligand binding activities based on the protein fold common to the LacI core and other protein families. Comprehensive understanding of LacI behavior and function may allow extension of design principles to generate even more complex allosteric systems.

描述（由申请人提供）：生物系统普遍采用结合/催化事件级联来传递有关环境和生理状态的信息。这种通讯网络需要通过变构机制改变蛋白质功能。这种分子内信号转导缺乏原子水平的洞察力，促使我们继续研究的“开关”途径，在研究乳糖阻遏蛋白（LacI）。在LacI功能中，配体结合信息必须流经广泛分离的诱导剂和DNA结合位点之间的蛋白质结构。我们的最终目标是一个原子水平的视图和连续的时间轴的构象变化，导致释放的操作DNA和随后的下游基因的转录。我们最近使用有针对性的分子动力学模拟（TMD）预测原子水平的变构途径，这是一致的大型图书馆的实验数据的LacI。再加上可用的表型信息，该模型提供了一个独特的框架，设计新的实验，以检查和重新设计的构象行为的LacI和评价TMD作为一个通用的工具，用于破译变构途径。实验提出了检查具体功能的LacI：（1）潜在的单一（2）核心N-亚结构域界面在采用交替构象中的作用;（3）连接LacI核心N-和C-亚结构域的3-链核心枢轴中的氨基酸对结构转变的调节;（4）对称性对变构反应的影响。位点特异性诱变将与详细的生物物理学分析（以及，如可能，结构数据）结合使用，以评估特定氨基酸在变构途径中的作用以及二聚体内不对称氨基酸变化的影响。LacI提供了一个独特的系统，具有必要的信息背景和工具，以严格测试TMD计算的预测。随着可靠性的建立，TMD可用于更充分地利用越来越多的变构蛋白质的终点结构。最后，在LacI变构变化的信息可以集成与最近成功设计的新的配体结合活性的基础上，常见的LacI核心和其他蛋白质家族的蛋白质折叠。对LacI行为和功能的全面理解可能允许扩展设计原理以产生更复杂的变构系统。