NMR Studies on Protein Dynamics and Its Relevance to Functional Kinetics.

Existence of a strong relationship between protein's dynamics and its functional kinetics was suggested decades ago. Even though different groups investigated this relationship, technical difficulties and lack of appropriate methodology make full understanding of this relationship quite challenging. In this project we approach investigation of this phenomenon from several different points. In the first part of the project, we demonstrate NMR based approach to elucidate lysine side-chain dynamics, as well as dynamics of hydrogen bonds in which they are involved. Such methodology provides us with detailed information on protein's behavior at atomic and pseudo-atomic resolutions. As amino acid side chains are involved in macromolecular interactions, knowledge on side-chain dynamics allows better understanding of the dynamic nature of such interactions. Presented data strongly suggests that hydrogen bonds involving lysine NH3+ groups have a very dynamic and transient nature. In the second part, we investigated the role of domain dynamics in translocation kinetics of human transcription factor Egr-1. Nature has designed Egr-1 in such a way, at the same time with strong DNA binding affinity for high target specificity, that it is rapid enough in translocation on the human genome to satisfy requirements of its biological functions. Our data strongly suggests that Egr-1 is able to achieve rapidity and high specificity due to the dynamic nature of its domains in the nonspecific complex with DNA. We also demonstrated that nature's way of designing zinc-finger transcription factors can be used to optimize activity of artificial ones. In the final part of this project, we demonstrated novel NMR based in situ method for elucidation of proteins' characteristics in various biological fluids. Using this method we determined oxidation speed of HMGB1 protein in human blood serum and saliva, as well as in extracellular fluids. Different effect of these fluids on HMGB1's oxidation clearly showed the importance of in situ experiments for better understanding of protein function in its natural environment. Studies presented here demonstrate that in order to understand the protein's dynamics/functional kinetics it is essential to simultaneously use different orthogonal approaches.