Research Project
10135620
Abstract. The research program of the Latham Laboratory centers on understanding the relationship between structure, dynamics, and function. We are particularly interested in examining this relationship in large macromolecular assemblies, which have for the most part been recalcitrant to detailed structural and dynamics studies. We couple high-resolution methyl-based nuclear magnetic resonance (NMR) spectroscopy, which is capable of probing macromolecules up to ~1 MDa in size, with other biophysical (e.g., lanthanide resonance energy transfer ? LRET) and biochemical techniques to better understand function. Currently, the focus of our program is on two protein complexes that bind to nucleic acids: the universally conserved and essential DNA double strand break repair complex Mre11-Rad50-Nbs1 (MRN) and the mRNA cleavage and poly-adenylation assembly cleavage stimulation factor complex (CstF). In both cases, existing structural and biochemical studies have suggested a role for protein motions in the function of the complex. However, many questions still remain about the interplay of structure and dynamics and how these relate to and control MRN and CstF activity. For the MRN complex, X-ray crystallography, cryo-electron microscopy, and small angle X-ray scattering studies have shown large structural changes within the complex upon ATP binding and have also hinted at local domain motions upon dsDNA binding; however, the timescale of these movements, other key structural states, their importance in function, and local dynamics have not been described. Similarly for the CstF complex, X-ray crystallography and NMR spectroscopy on isolated domains have given views of local structure and dynamics and provided a clue for nascent mRNA recognition; yet, the architecture of the full complex and complete structure of the RNA-bound state is missing. Additionally, disease associated mutations have been noted for both the MRN and CstF complexes. Thus, a main goal of the parent grant is to describe the structures and dynamics of these two nucleic acid binding complexes to better understand the how these characteristics affect function.
