Abstract
Spin-label electron spin resonance (ESR) spectroscopy has been extensively developed in the latest decade for studying problems in the fields of biology, physics, and chemistry. With the site-directed spin-labeling techniques, ESR can be employed to resolve the complexity of molecular dynamics, probing local environments of various molecular complexes such as protein, membrane, and macromolecular assemblies. In particular, continuous wave (cw) ESR and double electron-electron resonance (DEER) are among the most powerful ESR techniques. This dissertation demonstrates three biophysical applications of the ESR techniques that have never been reported. First, we describe how useful the ESR technique can be utilized to reveal details of molecular motions of spin-labeled biomolecules as confined in nanochannels. Specifically, we characterize the rotational dynamics of a long (14-residue) proline-based peptide (approximately 4 nm in length) under anisotropic nanoconfinement using spin-label ESR techniques as well as spectral simulations. We show by pulsed ESR experiments that the conformations of the peptide in several different nanochannels and a bulk solvent are retained. Parameters characterizing the dynamics of the peptide regarding temperature (200 ~ 300 K) and nanoconfinement are determined from nonlinear least-squares fits of theoretical calculations to the multifrequency (X- and Q-band) experimental spectra. Remarkably, we found that this long helical peptide undergoes a large degree of rotational anisotropy and orientational ordering inside the nanochannels, but not in the bulk solvent. The rotational anisotropy of the helical peptide barely changes with the nanoconfinement effects and remains substantial, as the nanochannel diameter is varied from 6.1 to 7.1 and 7.6 nm. This finding is advantageous for addressing purposes of anisotropic nanoconfinement and for advancing our understanding of the rotational dynamics of nano-objects as confined deeply inside the nanostructures of materials. In the second project presented in this dissertation, we report a ESR study of Bcl-2 associated X (BAX) protein. BAX protein plays a key role in the mitochondria-mediated apoptosis. However, it remains unclear by what mechanism BAX is triggered to initiate apoptosis. Here, we reveal the activation mechanism underlying the transformation from inactive to active BAX. An inactive BAX monomer was found to exhibit conformational heterogeneity and exist at equilibrium in two populations of conformation, one of which has never been reported. We show that upon apoptotic stimulus by BH3-only peptides, BAX can be induced to convert into either a ligand-bound monomer or an oligomer through a conformational selection mechanism. The kinetics of reaction is studied by means of time-resolved ESR, allowing a direct in-situ observation for the transformation of BAX from the native to the bound states. In vitro mitochondrial assays provide further discrimination between the proposed BAX states, thereby revealing a population-shift allosteric mechanism in the process. BAX′s apoptotic function is shown to critically depend on excursions between different structural conformations. In the third project, we apply the ESR techniques to investigate the effects of molecular crowding on protein stability. We carry out a comprehensive investigation on the conformational stability of T4 lysozyme (T4L) enzyme in varying crowding conditions, 300 − 500 g/L of crowders (including BSA protein, glycerol, Ficoll, and PVP polymers), using cw-ESR, circular dichroism, and Thermofluor spectroscopy methods. Double-labeled spectra were used to probe the local dynamical changes and distance distribution of T4L protein in the applied crowded and thermal conditions. ESR spectra were obtained from three T4L mutants to study the crowding effects on the tertiary structure (with mutant T4L-A), secondary structure (with mutant T4L-B), and hinge-bending activity (with mutant T4L-C) at temperatures 280 − 343 K. The results of the T4L-A and T4L-B show a decreased structural stability, in terms of conformational dynamics and free energy, with increasing concentration of the crowders. In contrast, the structural stability of the T4L-C mutant was found to increase with the crowder concentrations. This study indicates that structural domains or segments of a protein respond differently to molecular crowding effects. In summary, results presented in this dissertation have expanded the applications of spin-label ESR techniques one step further to resolving several important problems in the interdisciplinary field of biology, physics, and chemistry.