Proteins in pre-existing conformational equilibria sample different conformational states, some of which have important functional roles. Among intermediate conformational states, some proteins adopt a molten globule (MG) structure, which is compact and contains a relatively high content of native-like secondary structure, but has fewer tertiary contacts. The MG is a dynamic, flexible intermediate that adapts to a variety of conformations. Growing evidence that the MG is a hub in conformational equilibria between folded states emphasizes its putative functional importance. While the MG states of many α-helical proteins have been thoroughly studied, our understanding of β-sheet protein dynamics is incomplete. The plasticity of MG-like states of intestinal fatty acid binding protein (I-FABP), a β-barrel protein, and the T4 lysozyme (T4L) L99A mutant, a predominately α-helical protein, are thought to facilitate binding of a variety of ligands to their large cavities (230 and 150 �^3 for I-FABP and T4L L99A, respectively). X-ray crystal and nuclear magnetic resonance structures of I-FABP and T4L L99A do not identify an open conformation that permits ligand entry, suggesting that a rare MG may be facilitative.

Site-directed spin labeling and EPR spectroscopy (SDSL EPR) is a sensitive tool for identifying backbone dynamics, conformational exchange, and ligand binding of proteins. In this dissertation, a panel of EPR experiments (continuous wave ((CW)) EPR, saturation recovery, and double electron-electron resonance) provides information on nanosecond-microsecond timescale motions, their amplitudes, and the thermodynamic equilibrium between these states to lay a basis for the structure and dynamics of the I-FABP and T4L L99A MGs. These results are supported by other forms of optical spectroscopy (intrinsic fluorescence and dynamic light scattering).

As a means to populate rare intermediates, hydrostatic pressure is known to shift conformational equilibria to populate intermediate states through cavity hydration or structure-relaxation mechanisms. Internal hydration of proteins has structural parallels with the MG state. A comparison of the acid pH-stabilized MG of I-FABP and its high pressure states reveals structural similarity, in support of a model for protein conformational equilibria that involves a limited number of discrete conformational states rather than a continuum of intermediate conformations.

The MG-like character of the high pressure state of the T4 lysozyme cavity mutant L99A is also investigated using pressure-resolved CW EPR. In contrast with wild-type T4L, the L99A mutant shows site-specific smooth sigmoidal transitions with pressure that indicate a two-state equilibrium between the native and intermediate states. The goal of this work is to determine how cavity hydration affects the protein structure allosterically.

The main goal of this dissertation is to characterize the extent of structural heterogeneity of the MG from the SDSL EPR perspective, which encompasses fluctuation amplitudes, motional timescale, and the thermodynamic equilibrium state of the MG.

Proteins in solution are dynamic molecules that exhibit conformational flexibility across a range of time and length scales, and characterizing the functional role of protein motion is a primary goal in molecular biophysics. High hydrostatic pressure has emerged as a powerful probe of protein conformational flexibility. Development of instrumentation and methodologies that enable electron paramagnetic resonance (EPR) experiments on proteins at high pressure is the central aim of the work presented in this dissertation.

Pressurization of proteins reveals regions of elevated compressibility, and thus flexibility, within individual conformational states, but also shifts conformational equilibria such that “invisible” excited states become accessible for spectroscopic characterization. Current evidence indicates that pressure cleanly shifts the relative populations of states solely according to differences in partial molar volume without altering the shape of the conformational free energy landscape. Thus, variable pressure is a powerful tool for dissecting details of the landscape, and site-directed spin labeling coupled with electron paramagnetic resonance spectroscopy (SDSL EPR) is an ideal strategy in terms of sensitivity and time scale to detect the effects of pressure and interpret them in terms of structure and dynamics. In this dissertation, newly developed high-pressure instrumentation for both variable-pressure continuous-wave EPR and pressure-resolve double electron-electron resonance (PR DEER) of proteins in aqueous solution is described. The applications presented illustrate the considerable potential of the methods to: (1) identify compressible (flexible) regions in a folded protein; (2) determine thermodynamic parameters that relate conformational states in equilibrium; (3) populate and characterize excited states of proteins undetected at atmospheric pressure; (4) reveal the structural heterogeneity of conformational ensembles and provide distance constraints on the global structure of pressure-populated states. The SDSL EPR results are complemented by global secondary structure information provided by high-pressure circular dichroism experiments.

This work lays the foundation for future developments in high-pressure SDSL EPR, including pressure-jump relaxation spectroscopy to determine the lifetime of conformational states in the millisecond range and high pressure saturation recovery exchange spectroscopy to enable measurement of lifetimes of states in the microsecond range. SDSL EPR has unique advantages for the study of membrane proteins in their native environment under physiological conditions, and applications of high-pressure SDSL EPR to explore the conformational equilibria and dynamics of integral membrane proteins is a high priority for future work.

Phosphoryl transfer from a histidine kinase to a response regulator in two-component signaling systems leads to cellular modifications that enable bacteria to adapt to environmental changes. The response regulator NarL, of the Escherichia coli Nar two-component system, becomes phosphorylated by the histidine kinase NarX and regulates genes involved in nitrate respiration. Phosphorylation at the N-terminal "receiver" domain of NarL exposes distant molecular surfaces, including regions of the C-terminal domain that dimerize upon DNA binding. To investigate other alterations in domain surface interactions upon phosphorylation, NarL and its individual domains were examined by analytical ultracentrifugation. Phosphorylation was demonstrated to induce full-length NarL dimerization and tetramerization, and N-terminal domain dimerization. The C-terminal domain was unable to dimerize alone, suggesting that dimerization of the in-tact protein occurs via receiver domains, while C-terminal domain dimerization is driven by DNA binding. Independent receiver-domain dimerization implicates the widespread alpha4-beta5-alpha5 surface as the dimerization site, since this region is unavailable in full-length NarL, which was shown to be monomeric. Also, receiver domain dimerization and NarL oligomerization may fulfill binding requirements at low-affinity promoter regions.

Two unphosphorylated NarL crystal structures reveal, previously unreported, distinct equilibrium states with variability in residue positions and polar contacts. The conformations of two activation-associated residues in the receiver domain show that the monoclinic NarL crystal structure represents a semi-activated state. Both structures were analyzed with respect to the interdomain interface and active site in order to gain an understanding of the activation mechanism that leads to domain separation. A solvent-accessibility analysis, along with structural comparisons, confirmed that the conformation of Lys196, in the semi-activated NarL structure, is also representative of activation. Mobility ratios and correlation fluctuation calculations showed that vital interface-loop residues exhibit low mobilities, however can affect the motion of other interface residues, including those that bind DNA. Therefore, interface mutations that lead to constitutively active phenotypes may also result from the interdependence of residue motions. In contrast to the interface loops, the active-site loops were characterized by high mobility ratios. Structural modifications of these flexible loops are expected to coincide with the proposed movements of certain active-site residues.

Although many functionally important processes occur in the μs-ms timescale, few spectroscopic techniques can report direct dynamic information of biological systems in this time domain. One of these is SDSL-EPR (Site-Directed Spin Labeling Electron Paramagnetic Resonance). In the versatile toolbox of EPR methods, ST (Saturation Transfer)-EPR is uniquely capable of extending the motional-sensitivity range of EPR to as long as 10-3s, which is particularly attractive for studying protein dynamics. However, the method has not previously been generalized for the study of the internal dynamics of ordinary proteins. The development of methodologies that enable the ST-EPR technique to probe μs-ms internal protein dynamics under ambient conditions is the central aim of the work presented in this dissertation.The fast rotational diffusion of small soluble proteins and side chain internal motions limit the utility of ST-EPR to monitor conformational dynamics in the μs-ms range. To reduce the interference of these fast motions and extend the applicability of ST-EPR, we combine both stationary-phase techniques for protein immobilization and novel nitroxide spin labels with highly restricted internal motions. In all cases reported, the spin label is attached to a cysteine residue introduced by site-directed mutagenesis. The results from this work demonstrate that: (1) The applicability of SDSL ST-EPR for monitoring protein internal modes can, in general, be extended to ordinary soluble and membrane proteins via the use of a disulfide-linked bifunctional spin label side chain (RX) together with immobilization of the protein on a Sepharose solid support. Using the well-characterized proteins (T4 Lysozyme and Myoglobin), various contributions to the slow motions measured by RX and ST-EPR were dissected and the feasibility, versatility, and sensitivity of ST-EPR for monitoring protein internal dynamics were demonstrated. (2) To overcome potential perturbations to structure and dynamics caused by the cross-linking bifunctional RX side chain, two disulfide-linked spin label side chains attached at a single cysteine and having highly restricted internal motions were characterized and shown to be comparable to RX for the detection of slow protein motions. (3) Finally, a novel highly immobilized spin label side chain attached via a thioether linkage at a single cysteine was characterized. Unlike the disulfide-linked side chains, the new side chain, designated R9, reacts preferentially with solvent-exposed cysteine residues and is non-reducible by common reagents used in protein studies. R9 has great potential for describing the magnitude of structural changes and probing protein dynamics. Collectively, this work advances and broadens significantly the SDSL-EPR methodology for monitoring protein motions, making the direct measurement of internal dynamics possible within the important μs-ms time domain by providing new experimental tools and strategies. The results lay the foundation for the use of new spin labels in complex systems, including membrane proteins.

Proteins in solution exhibit structural fluctuations on a wide range of characteristic time scales, from fast backbone motions in the picosecond to nanosecond range to slow conformational exchange in the microsecond to millisecond time domain. These motions can play important roles in function, thus elucidation of molecular mechanisms underlying function requires experimental techniques capable of measuring motions on these time scales. Site directed spin labeling and EPR spectroscopy (SDSL-EPR) has been established as an important tool in protein science for studying structure, identifying dynamically disordered sequences, and monitoring conformational switching triggered by chemical or physical signals in soluble and membrane-bound proteins. However, it remains to be investigated whether fast backbone motions contribute to the spectra of the spin label side chain R1 in well-ordered sequences of regular secondary structure, and how slow conformational exchange between substates can be detected at equilibrium; these are goals of this dissertation.

The results from this work support a model wherein variations in the EPR spectral lineshape of R1-labeled proteins measures the amplitude of fast but constrained backbone fluctuations on the nanosecond time scale, and show that these motions are correlated with the local packing density. In addition, it is shown that osmolyte perturbation SDSL (OP-SDSL) can be used to identify conformational equilibria between substates that have characteristic lifetimes > 100 ns. The newly-developed OP-SDSL strategy was employed along with continuous wave (CW) lineshape analysis to map molecular flexibility of sperm whale myoglobin in folded and partially folded states and of core-repacking mutants of T4 lysozyme. For myoglobin, the results reproduce and extend earlier NMR studies, thus validating the OP-SDSL method and highlighting advantages of the inherent EPR time scale. For T4 lysozyme, it is found that core repacking mutations that generate internal cavities can give rise to new conformational substates in solution, despite the fact that earlier studies show that the corresponding crystal structures of WT and mutants were essentially identical. The results also show that ligand binding to some of the engineered cavities dramatically shifts the populations of substates towards the native-like state.

Collectively, the results of this research advance the SDSL-EPR methodology for facile mapping of protein flexibility over the important picosecond to millisecond time domain and demonstrate the utility of the technology for exploring the molecular basis of protein function.