NMR Studies on Kinetics of Processes Involving DNA-Binding Proteins

Macromolecular interactions do not just encompass the coming together of two or more macromolecules, but rather the speed of the interactions play a very important role in proper cellular function. In response to external stimuli, DNA binding proteins very efficiently carry out their cognate tasks by making use of different kinetic mechanisms. Here we quantitatively analyze the kinetics of various processes involving DNA binding proteins. Chapter I provides an introduction to the different macromolecular process under investigation and the role of kinetics in determining their function. The primary means of investigation of these macromolecular interactions in terms of structure and kinetics is done using nuclear magnetic resonance (NMR) spectroscopic methods. Chapter II provides the theoretical description of how NMR based methods enable us to analyze kinetics involving macromolecules occurring at different time scales. Firstly we develop a TROSY based z-exchange methodology to analyze the kinetics of translocation of a transcription factor, HoxD9 homeodomain, between two cognate DNA molecules in Chapter III. The facilitated target search process in a very challenging problem, which defines the function of different DNA binding proteins and the kinetic shortcuts taken by these macromolecules in speeding up the target location. In Chapter IV the NMR based methods used to dissect the roles of different translocation processes is extensively analyzed using simulations to verify their validity range. Here we also look into the effects of various microscopic events on the macroscopic rates measured using biophysical approaches. The redox regulatory proteins continually keep the cellular environment reductive and the redox state of DNA binding proteins such as HMGB1 could play an important role in its function. The reduction/oxidation kinetics of HMGB1 along with redox regulatory proteins are extensively analyzed in Chapter V using NMR based methods. This thesis provides an understanding into the physiologically relevant macromolecular interactions in terms of kinetics by the development of novel biophysical methods, simulations and experiments.