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Proteins are nature’s molecular machines. Over the last 40 years, the approach to understanding how they function has been based on knowing their precise three-dimensional structures.  More recently it has become clear that proteins undergo extensive fluctuations over a broad range of timescales and this is critical for their ability to bind biological ligands (or drugs), catalyze reactions, and somewhat ironically, fold into their three-dimensional structures. Thus, a static structural view of a protein provides a starting point for investigating its functional properties and leads directly to studying conformational dynamics.

The issue of protein dynamics has direct relevance to drug design. It is now clear that the dynamics of proteins affect the kinetics and thermodynamics of drug binding. This has made drug design and modeling particularly challenging as protein dynamics are complex and until now difficult to characterize. All these considerations are true even when just considering classical drug design where drugs are targeted to a protein’s active sight.  However, researchers now recognize a whole new class of so-called “allosteric drugs”; these drugs bind away from the active and exert their control at a distance. This leads to a general question:  How does ligand binding affect a protein throughout its entire structure? What is the physical basis for propagation of a binding event to distal regions of the protein? We are now pursuing these questions as a means to aid the a deeper understanding of target molecules (proteins) which will facilitate the development of allosteric drugs.  Our research will also have direct relevance to protein design and engineering, especially in the case of proteins designed to be drugs themselves.

The research in my laboratory is directed at the relationship between protein structure and dynamics and how these respond to perturbations such as binding (natural ligands or drugs) and mutation (evolution). NMR spectroscopy is an experimental tool uniquely suited to study both structure and dynamics in proteins and other biological macromolecules. A major advantage of NMR is that spectroscopic probes are distributed uniformly throughout the biomolecule, such as NH or CH atom pairs, providing large amounts of molecular information. We complement our experimental studies with molecular dynamics simulations, explicit simulation of protein, ligand, and solvent that evolve under an atomic-level force-field. This is done in collaboration with Jan Hermans in the Dept. of Biochemistry.

Research projects in the lab fall into two main categories:

(1)  The first is concerned with the biophysical nature of dynamic motions in enzymes and other proteins.  Even though dynamics are prevalent in all proteins, very little is known about dynamics when compared to the existing body of structural knowledge. Knowledge of the “moving parts” are likely to add considerable insight into phenomena such as catalysis, cooperativity, and allostery.  We are using 15N, 13C, and 2H NMR spin relaxation methods to monitor and characterize motions on the ps-ns timescale in enzymes and protein-ligand complexes.  We have begun our studies on a model globular protein, eglin c, a serine protease inhibitor, to test our methodology and answer general questions about motions in globular proteins. We are also working on several PDZ (PSD95 - Discs Large - ZO-1) domains, which are protein-protein recognition motifs found in signal transduction and synaptic complexes. The site-specific information obtained from NMR will gain value when coupled with traditional thermodynamic and kinetic information obtained through other biophysical measurements (CD, calorimetry, stopped-flow fluorescence, etc.), including MD simulations.

(2)  The second area of research is aimed at characterizing the structure and dynamics of proteins involved in DNA repair. Maintaining the integrity of genetic information is a high priority in the cell, as DNA damage is brought about by external insults and internally programmed cell biochemistry. DNA lesions not repaired accurately and expeditiously can leave mutations or deletions that become permanently integrated into the genome; in unfavorable cases this can lead to an inheritable predisposition to cancer. Therefore, genomic stability depends critically upon the protein components of the DNA repair machinery and associated signaling. As these components are identified, their structural biology will provide valuable details of the mechanisms of genome maintenance and cancer prevention.

Andrew Lee obtained his initial training in physical chemistry at Pomona College. As a graduate student of Dr. David E. Wemmer in biophysical chemistry at UC Berkeley, he focused on heteronuclear, multidimensional NMR spectroscopy as a method for structure determination of protein-RNA complexes. His postdoctoral work with Dr. A. Joshua Wand (University of Pennsylvania) yielded a series of studies which employed NMR relaxation measurements to probe the role of motional dynamics in protein stability and molecular recognition.