Quantitative Studies of the Biotin Regulatory System

生物素调节系统的定量研究

基本信息

批准号：
7915594
负责人：
DOROTHY BECKETT
金额：
$ 29.54万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-05-01 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7915594
关键词：
Acetyl-CoA Carboxylase Amino Acids Anabolism Bacteria Behavior Binding Binding Sites Biochemical Biotin Calorimetry Communication Complex Coupled DNA Binding Deuterium Exchange Measurement Development Diabetes Mellitus Dimerization Disease Enzymes Equilibrium Escherichia coli Evolution Fatty Acids Funding Gene Expression Genes Genetic Transcription Genome Glean Global Change Glucose Health Heart Diseases Homo Human In Vitro Kinetics Laboratories Lead Ligand Binding Ligands Ligase Malignant Neoplasms Mass Spectrum Analysis Measurement Measures Membrane Proteins Metabolic Metabolism Methods Molecular Nutrient Obesity Operon Organism Output Physiological Process Property Proteins Role Signal Transduction Signal Transduction Pathway Site Site-Directed Mutagenesis Specificity Structure System Testing Thermodynamics Time Titrations Transcription Initiation Transcription Repressor/Corepressor Transcriptional Regulation Vertebral column Yeasts base biotin carboxyl carrier protein gene repression in vivo insight promoter protein function protein protein interaction research study response sedimentation equilibrium small molecule transcription factor

项目摘要

A common mechanism utilized by organisms to respond to metabolic demands is through changes in transcription of specific genes. This response can occur through complex signal transduction processes or through simpler mechanisms involving binding of metabolites to transcription factors to regulate their DNA binding function. One paradigm for achieving communication of metabolic requirements to gene expression is through the direct use of metabolic enzymes in regulating gene transcription. In this proposal experiments are described to elucidate the mechanism by which the Escherichia coli biotin protein ligase, BirA, responds to the demand for biotin at the molecular level. BirA funnels biotin into metabolism by catalyzing its linkage to the biotin-dependent carboxylase, acetyl CoA carboxylase, and binds sequence specifically to the biotin operator sequence to regulate transcription of the biotin biosynthetic genes. The function of this regulatory circuit reflects several properties including allosteric activation by a small molecule, use of a single protein surface for multiple interactions and kinetic control of protein function. In the proposed studies the molecular mechanism of the BirA functional switch will be elucidated using combined biophysical and biochemical measurements. These include equilibrium measurements of ligand binding by Isothermal Titration Calorimetry and protein association by sedimentation equilibrium, measurements of H-D exchange coupled to mass spectrometry, and kinetic measurements of protein association. The combined studies will reveal the basic molecular and cellular mechanisms of communication between metabolism and transcription.

生物体响应代谢需求的常见机制是通过特定基因转录的变化。这种反应可以通过复杂的信号发生转导过程或通过涉及代谢物结合的更简单的机制转录因子调节其 DNA 结合功能。实现的一种范例代谢需求与基因表达的沟通是通过直接使用调节基因转录的代谢酶。在这个提案中，实验被描述为阐明大肠杆菌生物素蛋白连接酶 BirA 响应的机制分子水平上对生物素的需求。 BirA 通过催化生物素将其引入新陈代谢与生物素依赖性羧化酶、乙酰辅酶A羧化酶连接，并结合序列专门针对生物素操纵序列来调节生物素生物合成的转录基因。该调节电路的功能反映了多种特性，包括变构通过小分子激活，使用单个蛋白质表面进行多种相互作用和动力学蛋白质功能的控制。在拟议的研究中，BirA 的分子机制将通过结合生物物理和生化测量来阐明功能开关。其中包括通过等温滴定量热法进行配体结合的平衡测量和通过沉降平衡进行的蛋白质关联，H-D 交换的测量耦合到质谱分析和蛋白质缔合的动力学测量。合并研究将揭示新陈代谢和细胞之间通讯的基本分子和细胞机制转录。