Optimization Of Nuclear Magnetic Resonance Spectroscopy Methods For Measuring Protein Hydration In Reverse Micelles

Water is fundamental to all aspects of protein function including folding, stability, catalysis, and dynamics. The unique characteristics of water make it the ideal solvent for supporting life but also make it incredibly difficult to study. While much has been learned about the role of water in protein function, a site resolved understanding of these interactions has remained elusive thereby leaving a large hole in the biophysical puzzle. Experimental techniques that provide a site-resolved view of protein hydration without mutation of the protein are necessary to understand the thermodynamic role of water on protein function. It has been shown that the combination of Nuclear Magnetic Resonance (NMR) spectroscopy and encapsulation of proteins within the core of reverse micelles can satisfy these requirements. The goal of this thesis is to apply and expand upon currently established techniques to make it generally applicable to all protein systems. First, previously established methods were used to examine the internal hydration patterns of staphylococcal nuclease variants with internal ionizable groups. The results demonstrate that water penetrates the hydrophobic core to stabilize buried ionizable groups. This study illustrates the utility of NMR detected hydration measurements for longstanding biophysical questions. Next, two methods to reduce data collection times were implemented for hydration dynamics measurements. This necessary time savings provides a platform for assessing the reproducibility and precision of NMR derived hydration measurements. A new data fitting method that allows for quantitative hydration dynamics measurements of protein regions generally contaminated by hydrogen exchange is introduced. Finally, one of the remaining artifacts associated with hydration dynamics measurements detected by NMR is addressed: hydrogen exchange relayed artifacts. This was accomplished by developing experiments to decouple the relaying spin and applying a new data fitting method. These methods allow for the first site and time resolved study of protein hydration in the absence of artifact is presented. These techniques introduced were applied to Ubiquitin encapsulated in AOT reverse micelles. The majority of slowed waters reside in concave regions of the protein surface. This suggests surface curvature is one of the contributing factors for the slowing of hydration waters. The experiments presented demonstrate the utility of using NMR for measuring protein-water interactions. The work presented expands and improves upon existing methodologies and provides a framework for artifact free site resolved measurement of protein water-interactions in all protein systems

Reverse Micelle Encapsulation And Its Use In Examining The Interplay Between Hydration And Protein Dynamics

As the universal solvent, water is unquestionably essential to most aspects of protein biophysics from protein folding to enzymatic activity. Much has been learned about the relationship between proteins and surrounding solvent waters; however, it is often difficult to experimentally examine these interactions in a site-specific manner without perturbing molecular structure. Furthermore, the effect of nearby hydration dynamics on protein dynamics (and, in effect, protein conformational entropy) is poorly understood at atomic resolution. With the use of a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and protein reverse micelle (RM) encapsulation, it is possible to examine both the dynamic behavior of waters in the protein hydration layer as well as protein dynamics for the same sample without physically altering the protein. The goal of this work is to use these complementary techniques in order to better understand the interplay between hydration and protein dynamics. First, we demonstrate the utility of NMR spectroscopy in monitoring and controlling the pH of the aqueous interior of reverse micelle ensembles. This leads to the ability to reliably confirm sample pH and structural fidelity upon RM encapsulation which is often difficult to accomplish using other techniques. Next we propose a novel approach to collecting and analyzing NMR hydration dynamics experiments with the use of non-uniform sampling (NUS) and nuclear Overhauser effect (NOE) mixing time buildup experiments. We examine factors contributing to the reproducibility and reliability of hydration ratios. Using these NOE-based hydration experiments, we then examine the hydration dynamics of hen egg-white lysozyme (HEWL) with and without a bound inhibitor. We find minimal retardation of hydration dynamics within a partially hydrophilic binding cleft; we detect waters within an internal pocket which are relatively fast; and we inspect trapped interfacial waters upon ligand binding. Finally, we use RM encapsulation to examine the effect of changes in solvent dynamics on fast (ps-ns) protein dynamics. While retardation of hydration dynamics seems to affect dynamics of aromatic side chains, it has little to no effect on other fast protein dynamics effectively confirming that protein conformational entropy is not slaved to solvent. This work represents a large leap forward in our understanding of the relationship between proteins and their hydrating environment

Protein Dynamics and Entropy: Implications for Protein-Ligand Binding

The nature of macromolecular interactions has been an area of deep interest for understanding many facets of biology. While a great deal of insight has been gained from structural knowledge, the contribution of protein dynamics to macromolecular interactions is not fully appreciated. This plays out from a thermodynamic perspective as the conformational entropy. The role of conformational entropy in macromolecular interactions has been difficult to address experimentally. Recently, an empirical calibration has been developed to quantify the conformational entropy of proteins using solution NMR relaxation methods. This method has been demonstrated in two distinct protein systems. The goal of this work is to expand this calibration to assess whether conformational entropy can be effectively quantified from NMR-derived protein dynamics. First, we demonstrate that NMR dynamics do not correlate well between the solid and solution states, suggesting that the relationship between the conformational entropy of proteins is limited to solution state-derived NMR dynamics. We hypothesize that this may be partially due to the role of hydration of the protein in its dynamics. Next, we expand our empirical calibration to over 30 distinct protein systems and demonstrated that the relationship between NMR dynamics and conformational entropy is both robust and general. Furthermore, we demonstrate that conformational entropy plays a significant role in macromolecular interactions. Using our empirical calibration, we then look to address if conformational entropy could be an important contribution to drug design. The latter process is often a brute force approach, and subsequent optimization of initial drug candidates is often a guess and check process. In silico drug design was thought to offer a more efficient and rational approach, but often relies on static structures. This minimizes or completely neglects the role that conformational entropy may play in binding. Here we experimentally determine the role of conformational entropy in the drug target p38a MAPK in binding to two potent inhibitors. We demonstrate evidence that conformational entropy may represent a tunable parameter in affinity optimization of lead compounds. This has important implications for lead optimization and strongly suggests that the role of conformational entropy be considered in drug design efforts