UCLA

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.