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G-protein–coupled receptors are the largest superfamily in the human genome, and involved in critical cellular signaling processes in living cells. Protein structural fluctuations are the key for GPCR function that is driven and modulated by a variety of factors that are not well understood. This dissertation focusses on understanding the activation of GPCRs using the visual receptor, rhodopsin as the prototype. Rhodopsin is an ideal candidate for this study, as it represents the largest class of GPCRs, and is known to demonstrate more noticeable structural changes upon activation compared to the other GPCRs. What structural fluctuations occur, the role of water, and how the retinal cofactor regulates the protein dynamics during rhodopsin activation are specific research problems addressed in this work. Hypothesizing an ensemble activation mechanism, experiments were conducted using a variety of techniques to probe structural and dynamical fluctuations of rhodopsin in native membranes, as well as in membrane mimetics such as detergent micelles. Time-resolved wide-angle X-ray scattering (TR-WAXS), small-angle neutron scattering (SANS), quasielastic neutron scattering (QENS), and electronic spectroscopy are among the prominent techniques used to gain insights into the photo-intermediates that are key to understanding the rhodopsin activation process. The small-angle neutron scattering (SANS) experiments revealed a volumetric expansion of the protein molecule upon photoactivation of rhodopsin. Electronic spectroscopy together with the differential hydration study revealed the crucial role of water in rhodopsin signaling process and signal amplification by water. The quasielastic neutron scattering study conducted on powdered rhodopsin probed the changes in the local dynamics that are regulated by the retinal cofactor of the rhodopsin molecule. The increased local steric crowding in the ligand-free opsin is consistent with collapsing of the apoprotein structure in the absence of the retinal chromophore leading to inactive opsin conformation. Finally, a time-resolved wide-angle X-ray scattering study was conducted using the X-ray free electron laser at the SLAC national laboratory to probe the early structural fluctuations in rhodopsin photoactivation. The preliminary pump-probe experiments conducted on rhodopsin in CHAPS detergent micelles revealed a light-triggered protein quake that occurs during the early activation stages of rhodopsin photoactivation. Thus the protein fluctuations underlying the GPCR function are revealed by neutrons, X-rays, and other photons in a combined implementation of both spectroscopic and scattering techniques as applied to the investigation of rhodopsin activation.

Self-assembly and the induced orientation of microscopic biological systems is of great scientific interest, because it holds the promise of many pharmaceutical applications. This dissertation presents experimental studies done on proteins, short DNA fragments, and cholesterol structures self-assembled in an aqueous environment. The goal is to probe the thermo-physical properties of these systems, their phases and phase transitions, in order to better under-stand the principles behind their unique assemblies and function. It is accepted that in all these systems the solvent water plays an important role on the assembly folding, orientation, and activity of biopolymers. However, the abundance of water in typical samples presents many experimental challenges. It is indeed the case that changes in the properties of hydration in watery environments are responsible for the dynamics of protein and DNA biomolecules. We have explored in more detail the thermodynamics, the structural properties, and the dynamics near structural transitions of biomolecules in their native aqueous environment.

This thesis presents an effort to understand the C-H-C transfer in enzymatic reactions from the comparison of different variants of enzymes that have unrelated protein sequences and structures, but catalyze the same chemical transformation. I evaluated the kinetic isotope effects (KIEs) and their temperature dependences and interpreted the findings in accordance with Marcus-like models. The enzyme system studied is dihydrofolate reductase (DHFR), which catalyzes the reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F) using reduced β-nicotinamide adenine dinucleotide 2' phosphate (NADPH) as a reducing agent. H-transfer reactions in typical enzymes from three genetically unrelated families, E. coli chromosomal DHFR (cDHFR, FolA), plasmid coded R67 DHFR (FolB), and pteridine reductase 1 (PTR1, FolM) were comparatively investigated. Chapter I provides a brief introduction to the thesis. Chapter II presents optimized procedures for a one-pot, enzymatic microscale synthesis of several NADPH isotopologues used in KIE experiments. Chapter III focuses on the application of novel competitive primary H/D KIE determinations. Chapter IV compares the H-transfer reactions between primitive R67 DHFR and the chromosomal DHFR, and Chapter V describes the investigation of H-transfer reactions at high and low ionic strengths with theoretical and experimental approaches in order to understand the unusual enhancement in H-transfer rate of R67 DHFR with increasing ionic strength. Chapter VI discusses an improved PTR1 purification procedure and comparisons of steady state kinetic parameters using PTR1 and cDHFR with H2F and dihydrobiopterin (H2B) substrates. Thus, the investigation of the H-transfer reaction catalyzed by cDHFR with an unnatural substrate, H2B is described. Finally, a summary is provided and future directions are discussed in Chapter VII.

Water has long been assumed to be essential for biological function. To understand the molecular basis of the role of water in protein function, several studies have established a correlation between enzyme activity and hydration level. While a threshold of hydration of 0.2 h (grams of water per gram of dried protein) is usually accepted for the onset of enzyme activity, recent works show that enzyme activity is possible at water contents as low as 0.03 h (Lind et al., 2004). Diffusion limitation in these experiments was avoided by monitoring enzyme-catalyzed hydrolysis of gas-phase esters. However, since water is also a substrate for the enzyme used in these experiments, they cannot be used to probe the possibility of activity at zero hydration. However, the pig liver esterase and C. rugosa lipase B are able to catalyse alcoholysis reactions in which an acyl group is transferred between an ester and an alcohol. Therefore, by following this reaction and using a gas phase catalytic system, we have been able to show that activity can occur at 0 g/g. These results led to the question of the accuracy of determinations of very low water concentrations; i.e., how dry is 0 g/g? Although gravimetric measurements of the hydration level do not allow us to define the anhydrous state of the protein with sufficient sensitivity, using 18O-labeled water, we have been able to quantify the small number of water molecules bound to the protein after drying, using a modification of the method of Dolman et al. (1997). Testing different drying methods, we have been able to determine a level of hydration as low as 2 moles of water per mole of protein (equivalent to 0.0006 h in the case of pig liver esterase) and have shown that in the case of the pig liver esterase, activity can occur at this hydration level. At the molecular level, if the hydration level affects activity, we can expect an effect on the protein dynamics. Neutron scattering spectra of hydrated powders, for instance, show that diffusive motions of the protein increase with the hydration (Kurkal et al., 2005) To address the question of the protein motions involved in the onset of enzyme activity at low hydration, we performed neutron scattering experiments on a pico-second time scale on dried powders. Preliminary results show a dynamical transition at hydration levels as low as 3 h. Molecular dynamic simulations have also been used in this study to access the dynamics of the active site. Overall, the results here show that pig liver esterase can function at zero hydration, or as close to zero hydration as current methods allow us to determine. Since the experimental methodology restricts this work to a small number of enzymes, it is unlikely that it will ever be possible to determine if all enzymes can function in the anhydrous state: however, the results here indicate that water is not an obligatory requirement for enzyme function.

Tyrosine hydroxylase (TyrH) catalyzes the rate-limiting step in the synthesis of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Phosphorylation of Ser40 of rat TyrH activates the enzyme by decreasing the affinity for catecholamines. In humans, there are four different TyrH isoforms with varying lengths for the regulatory domain. DOPA and dopamine binding studies were performed on the phosphorylated and unphosphorylated human isoforms. The Kd for DOPA was increased two times upon phosphorylation of hTyrH1, but no change was seen for hTyrH4; the Kd value decreased with the increase in the size of regulatory domain. The small effect on the Kd value for DOPA upon phosphorylation of hTyrH suggests that DOPA does not regulate the activity of hTyrH. Dopamine binds very tightly and upon phosphorylation the affinity for dopamine is decreased. This Kd value decreases with the increase in the length of the regulatory domain. The crystal structures of substrate complexes of the homologous enzyme phenylalanine hydroxylase (PheH) show a large movement of a surface loop (residues 131-155) upon amino acid binding. The corresponding loop residues (175-200) in TyrH play an important role in DOPA formation. This conformational change in TyrH loop was studied with fluorescence anisotropy. Three tryptophan residues in the TyrH, at positions 166, 233, and 372, were mutated to phenylalanine, and Phe184 was mutated to tryptophan. An increase in anisotropy was observed in the presence of phenylalanine and 6-methyl-5-deazatetrahydropterin (6M5DPH4), but the magnitude of the change of anisotropy with 6M5DPH4 was greater than that with phenylalanine. Further characterization of the sole tryptophan in the loop showed a decrease in the amplitude of the local motion only in the presence of 6M5DPH4 alone. The conformational change in wild type TyrH was examined by H/D exchange LC/MS spectroscopy in the presence of the natural ligands. Time-course dependent deuterium incorporation into the loop in the presence of ligands indicated that the pterin alone can induce the conformational change in the loop irrespective of whether iron is reduced or oxidized. From these results, one can conclude that the loop undergoes a conformational change upon pterin binding, making the active site better for amino acid binding.