Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

Princeton University, Princeton NJ

Investigators

Abstract

The molecular mechanisms by which small-molecule allosteric effectors activate gene-regulatory proteins for specific DNA binding are largely unknown. Understanding these mechanisms should provide general insights into molecular recognition, and inform our understanding of the biology of the molecules involved in these processes. The overall goal of the present work is to achieve an understanding of allosteric activation that accounts for both structural and energetic features of ligand binding and explains DNA specificity in the arginine repressor system of E. coli, ArgR. A combined biochemical and biophysical approach will be used to establish the quantitative foundations for a comprehensive understanding of the molecular and biological significance of ArgR-ligand interactions. The cornerstone of this approach is careful measurement of ligand-binding equilibria including determination of affinity, stoichiometry, and cooperativity. The goal in making these measurements is to define the ligand-occupancy states of the protein as a function of ligand concentration. This information is then used to set the conditions for studies of protein function and structure over the range of relevant ligand-occupancy states. This project has four aims. 1. Quantitative analysis of L-arg binding to ArgR using isothermal titration, analytical ultracentrifugation, and NMR. 2. Analysis of the effects of ligand-occupancy state on ArgR function in transcription and recombination. 3. Evaluation of changes in structure and dynamics as a function of ligand-occupancy state, using proteolytic cleavage and NMR. 4. Examination of ATP binding to evaluate whether this ligand confers functional or structural consequences for ArgR. These biochemical and biophysical experiments should provide the quantitative foundations for understanding the molecular and biological mechanisms of ArgR function, and should help to bring the functional and structural pictures of ArgR allosteric activation into a common focus. Biomolecules have difficult jobs to do. Many of them must recognize one ligand from an intracellular sea of similar ligands, and respond in a physiologically appropriate way. The range of molecular strategies used to execute complex cellular functions is probably very broad, but few examples have been fully characterized. An in-depth understanding of molecular strategies is fundamental to many areas of chemistry and biology. This research is aimed at expanding our general understanding of the mechanisms used by proteins to recognize and respond to small ligands that can potentiate their function.