Mapping molecular flexibility of spin labeled proteins on the nanosecond and longer time scales via CW lineshape analysis and osmolyte-perturbation EPR

• Main Author: López, Carlos Javier
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: eScholarship, University of California 2012
• Subjects: Biochemistry; Biophysics; DEER; EPR; myoglobin; osmolytes; Site-directed spin labeling; T4 lysozyme; Sperm whale
• Online Access: http://www.escholarship.org/uc/item/5r00c82c; http://n2t.net/ark:/13030/m5r2125b

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.