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/催化事件的级联来传输有关环境和生理状态的信息。该通信网络要求通过变构机制改变蛋白质功能。原子水平对这种分子内信号转导的洞察力的洞察力激发了我们对良好研究的乳糖抑制剂蛋白（LACI）中“开关”途径的持续研究。在LACI功能中，配体结合信息必须在广泛分离的诱导剂和DNA结合位点之间流过蛋白质结构。我们的最终目标是构象变化的原子级视图和顺序时间表，导致算子DNA的释放以及随之而来的下游基因的转录。我们最近使用靶向分子动力学模拟（TMD）来预测与LACI的大量实验数据库一致的原子级变构途径。加上可用的表型信息，该模型为设计新颖的实验提供了一个独特的框架，以检查和重新设计LACI的构象行为，并评估TMD作为解密变构途径的一般工具。提出了实验来检查LACI的特定特征：（1）单个（或仅几个）氨基酸的潜力触发诱导剂的变构反应；（2）核心n-辅助剂界面在采用替代构象中的作用；（3）在连接LACI核心的N-和C-亚群的3链核心枢轴中对氨基酸的结构转移调节；（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分析（以及可能的结构数据）一起使用，以评估特异性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，具有必要的信息和工具背景，以严格测试TMD计算的预测。有了既定的可靠性，TMD可用于更充分利用可用于变构蛋白的端点结构的增加。最后，基于LACI核心和其他蛋白质家族共有的蛋白质折叠的新型配体结合活性的新型配体结合活性的设计，可以将有关LACI的变构变化的信息整合在一起。对LACI行为和功能的全面了解可能会使设计原理的扩展能够产生更复杂的变构系统。