Solid-State NMR methods and techniques to characterize large membrane protein complexes



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In this dissertation I used existing Solid-State NMR and, I developed new techniques to study membrane proteins. All the studies and method development were demonstrated on the inward rectifier K+ channel KirBac1.1 found in Burkholderia pseudomallei. KirBac1.1 is homologous to human Kir channels, sharing a nearly identical fold. Like many existing Kir channel crystal structures, the 1p7b crystal structure is incomplete, missing 85 out of 333 residues, including the N-terminus and C-terminus. The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, I examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (w/w). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. With expressing KirBac1.1 in a U-15N,13C media and the usage of multidimensional Solid State Nuclear Magnetic Resonance (SSNMR) spectroscopy experiments I assigned chemical shift of 301 residues out of 333 residues, which revealed two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlights conformational changes associated with an open activation gate and suggest a unique allosteric pathway which ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. I also identify specific residues involved in this conformational exchange which are highly conserved among human Kir channels. Membrane proteins and lipids coevolved to yield unique co-regulatory mechanisms. Inward- rectifier K+ (Kir) channels are often activated by anionic lipids endemic to their native membranes and require accessible water along their K+ conductance pathway. To better understand Kir channel activation, we target multiple mutants of the Kir channel KirBac1.1 via solid state Nuclear Magnetic Resonance (SSNMR) spectroscopy, potassium efflux assays, and Förster- resonance-energy-transfer (FRET) measurements. In the I131C stability mutant (SM), we observe an open-active channel in the presence of anionic lipids with greater activity upon addition of cardiolipin (CL). Introducing three R to Q mutations (R49/151/153Q (TQ)) renders the protein inactive within the same activating lipid environment. Our SSNMR experiments reveal a stark reduction of lipid-protein interactions in the TQ mutant explaining the dramatic loss of channel activity. Water-edited SSNMR experiments further determined the TQ mutant possesses greater overall solvent exposure in comparison to wild type, but with reduced water accessibility along the ion conduction pathway, consistent with the closed state of the channel. These experiments also suggest water is proximal to the selectivity filter of KirBac1.1 in the open-activated state, but that it may not directly enter the selectivity filter. Our findings suggest lipid binding initiates a concerted rotation of the cytoplasmic domain subunits, which is stabilized by multiple inter-subunit salt bridges. This action buries ionic side chains away from the bulk water, while allowing water greater access to the K+ conduction pathway. This work highlights universal membrane protein motifs, including lipid-protein interactions, domain rearrangement, and water-mediated diffusion mechanisms. Up to this point, membrane protein water accessibility surface area is often investigated as a topological function via solid-state NMR. Here I leverage water-edited solid-state NMR measurements in simulated annealing calculations to refine structure. I measure solid-state NMR water proximity information and use this for refinement of KirBac1.1 using the Xplor-NIH structure determination program. Along with predicted dihedral angles and sparse intra- and inter-subunit distances, we refined the residues 1-301 to atomic resolution. All structural quality metrics indicate these restraints are a powerful way forward to solve high quality structures of membrane proteins using NMR.

Embargo status: Restricted until 01/2025. To request the author grant access, click on the PDF link to the left.



Solid State nuclear magnetic resoance (SSNMR), Membrane protein, Water edited spectroscopy