Abstract
The dynamic protein-protein and protein-ligand interactions of integral bitopic membrane proteins with a single membrane-spanning helix play a plethora of vital roles in the cellular processes associated with human health and diseases, including signaling and enzymatic catalysis. While an increasing number of high-resolution structural studies of membrane proteins have successfully manifested an in-depth understanding of their biological functions, intact membrane-bound bitopic protein-protein complexes pose tremendous challenges for structural studies by crystallography or solution NMR spectroscopy. Therefore, there is a growing interest in developing approaches to investigate the functional interactions of bitopic membrane proteins embedded in lipid bilayers at atomic-level. Here we demonstrate the feasibility of dynamic nuclear polarization (DNP) magic-angle-spinning NMR techniques, along with a judiciously designed stable isotope labeling scheme, to measure atomistic-resolution transmembrane-transmembrane interactions of full-length mammalian ~72-kDa cytochrome P450-cytochrome b5 complex in lipid bilayers. Additionally, the DNP sensitivity-enhanced two-dimensional 13C/13C chemical shift correlations via proton driven spin diffusion provided distance constraints to characterize protein-lipid interactions and revealed the transmembrane topology of cytochrome b5. The results reported in this study would pave ways for high-resolution structural and topological investigations of membrane-bound full-length bitopic protein complexes under physiological conditions.
Highlights
The dynamic protein-protein and protein-ligand interactions of integral bitopic membrane proteins with a single membrane-spanning helix play a plethora of vital roles in the cellular processes associated with human health and diseases, including signaling and enzymatic catalysis
We demonstrate the use of state-of-the-art sensitivity enhancement by dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) spectroscopy under magic angle spinning (MAS)[54,55,56,57,58,59,60,61,62] and a judiciously designed isotope labeling scheme to overcome the difficulties associated with the structural studies of full-length bitopic proteins
Our previous studies revealed that multidimensional MAS solid-state NMR (ssNMR) experiments performed on uniformly 13C-labeled cytochrome b5 reconstituted in DMPC mutilamellar vesicles (MLVs) gave rise to spectra with low signal-to-noise ratio at 310 K69
Summary
The dynamic protein-protein and protein-ligand interactions of integral bitopic membrane proteins with a single membrane-spanning helix play a plethora of vital roles in the cellular processes associated with human health and diseases, including signaling and enzymatic catalysis. We demonstrate the use of state-of-the-art sensitivity enhancement by dynamic nuclear polarization (DNP) NMR spectroscopy under magic angle spinning (MAS)[54,55,56,57,58,59,60,61,62] and a judiciously designed isotope labeling scheme to overcome the difficulties associated with the structural studies of full-length bitopic proteins The combination of these developments have enabled us to report the DNP solid-state NMR spectroscopy probing transmembrane-transmembrane interactions of the cytochrome P450-cytochrome b5 complex embedded in lipid bilayers at an atomic-level for the first time
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