UCLA

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.