Spectroscopic Studies of Membrane Protein Structure, Dynamics, and Function

Biomolecular NMR spectroscopy and EPR spectroscopy are established methods for investigating the structure and dynamics of membrane proteins. However, there are various experimental challenges in working with membrane proteins that has hindered the progress of membrane protein investigations including structure determination. One of the primary bottlenecks is the selection of a membrane mimic that will stabilize a functional membrane protein fold. Mimic selection is largely empirical, and only a select few detergents have led to NMR membrane protein structure determination. NMR spectra of membrane proteins in different detergents are extremely variable in quality and often do not lead to structure determination. Using two eight stranded β-barrel model systems, Opa50 and Opa60, solution conditions that influence the NMR spectra were systematically investigated in order to gain an understanding of the interactions between the bilayer mimic and membrane protein that stabilize a fold. Detergent and ionic strength had a significant impact on the quality of NMR spectra due to interactions between the extracellular loops within a monomeric protein-detergent complex. DMPC lipid nanodiscs were also used as a bilayer mimic, and the resulting fold was comparable to the micelle embedded structure.
In addition to structure, magnetic resonance investigations of membrane proteins can also provide information about membrane protein dynamics. The dynamics of model helical membrane protein, TM0026 were investigated using EPR spectroscopy and NMR relaxation. We have noted a correlation between EPR scaled mobility and inverse second moment data with 15N NMR relaxation data. Both the NMR and EPR data indicate that both methods can reflect membrane protein backbone dynamics in the ns timescale. In the case of TM0026, a proline kink decouples the dynamics of the transmembrane helical backbone such that the N-terminal region of the helix is more dynamic than the C-terminal region. The results provide evidence that although the EPR lineshapes and interactions of the nitroxide are different in membrane proteins the lineshapes reflect backbone dynamics in the ns time regime.