Applications of Site-directed Spin Labeling Electron Paramagnetic Resonance to Studying the Structure, Dynamics and Interactions of Membrane Proteins

Freed, Daniel Marlay, Department of Chemistry, University of Virginia
Cafiso, Dave, Department of Chemistry, University of Virginia
Landers, James, Department of Chemistry, University of Virginia
Venton, Jill, Department of Chemistry, University of Virginia
Nakamoto, Robert, Department of Molecular Phys and Biological Physics, University of Virginia

Site - directed spin labeling (SDSL) electron paramagnetic resonance (EPR) is especially well - suited for the notoriously difficult analysis of membrane proteins, and offers numerous important advantages compared to more traditionally used techniques. Of these, arguably the most important is the ability to study membrane proteins in a more physiologically relevant environment. The first part of the following work describes the use of SDSL - EPR in conjunction with X - ray crystallography to study the substrateinduced unfolding of the Ton box motif in BtuB, the Escherichia coli outer membrane TonB - dependent corrinoid transporter. The data indicate that, compared to when BtuB is reconstituted in lipid bilayers, the Ton box equilibrium is shifted towards the folded state by 3 kcal/mol in BtuB crystals, with equal contributions attributed to both the crystal lattice and osmolytes in the crystallization buffer. Although these results fortify the notion that SDSL - EPR is a valuable tool for studying membrane proteins, a better understanding of spin label energetics - within the context of the local protein environment - is essential for the unambiguous interpretation of EPR spectra and the use of long - range distance restraints obtained from spin labels. Towards that end, the molecular origins of EPR spectra from hydrocarbon - exposed sites on BtuB were examined using various EPR techniques combined with X - ray crystallography and quantitative modeling. Collectively, the data indicate that at such sites, spin label configuration and dynamics are influenced by the solvation environment iii at the protein - hydrocarbon interface, and that this environment modulates weak interactions of the spin label with the protein surface. Finally, substrate - mediated transmembrane signaling by active membrane transporters and their interaction with the energy - coupling protein TonB was examined. The effects of substrate, transport - defective Ton box mutations and a competing substrate for BtuB on the affinity of the interaction were characterized. Furthermore, the structure of TonB in solution and bound to the transporters was examined using SDSL - EPR, and from this collective body of data a transport mechanism is proposed that accounts for the findings. The results presented have important implications for the design of novel antibiotics that interfere with key intermolecular interactions made by TonB.

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PHD (Doctor of Philosophy)
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