Solid-State NMR of Membrane Proteins in Lipid Bilayers: To Spin or Not To Spin?

Abstract

Metrics & MoreArticle Recommendations CONSPECTUS: Membrane proteins mediate a plethora of cellular functions and represent important targets for drug development. Unlike soluble proteins, membrane proteins require native-like environments to fold correctly and be active. Therefore, modern structural biology techniques have aimed to determine the structure and dynamics of these membrane proteins at physiological temperature and in liquid crystalline lipid bilayers. With the flourishing of new NMR methodologies and improvements in sample preparations, magic angle spinning (MAS) and oriented sample solid-state NMR (OS-ssNMR) spectroscopy of membrane proteins is experiencing a new renaissance. Born as antagonistic approaches, these techniques nowadays offer complementary information on the structural topology and dynamics of membrane proteins reconstituted in lipid membranes. By spinning biosolid samples at the magic angle (θ = 54.7°), MAS NMR experiments remove the intrinsic anisotropy of the NMR interactions, increasing spectral resolution. Internuclear spin interactions (spin exchange) are reintroduced by RF pulses, providing distances and torsion angles to determine secondary, tertiary, and quaternary structures of membrane proteins. OS-ssNMR, on the other hand, directly detects anisotropic NMR parameters such as dipolar couplings (DC) and anisotropic chemical shifts (CS), providing orientational constraints to determine the architecture (i.e., topology) of membrane proteins relative to the lipid membrane. Defining the orientation of membrane proteins and their interactions with lipid membranes is of paramount importance since lipid−protein interactions can shape membrane protein conformations and ultimately define their functional states.In this Account, we report selected studies from our group integrating MAS and OS-ssNMR techniques to give a comprehensive view of the biological processes occurring at cellular membranes. We focus on the main experiments for both techniques, with an emphasis on new implementation to increase both sensitivity and spectral resolution. We also describe how the structural constraints derived from both isotropic and anisotropic NMR parameters are integrated into dynamic structural modeling using replica-averaged orientational-restrained molecular dynamics simulations (RAOR-MD). We showcase small membrane proteins that are involved in Ca 2+ transport and regulate cardiac and skeletal muscle contractility: phospholamban (PLN, 6 kDa), sarcolipin (SLN, 4 kDa), and DWORF (4 kDa). We summarize our results for the structures of these polypeptides free and in complex with the sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA, 110 kDa). Additionally, we illustrate the progress toward the determination of the structural topology of a six transmembrane protein associated with succinate and acetate transport (SatP, hexamer 120 kDa). From these examples, the integrated MAS and OS-ssNMR approach, in combination with modern computational methods, emerges as a way to overcome the challenges posed by...

Nothing Regular about the Regulins: Distinct Functional Properties of SERCA Transmembrane Peptide Regulatory Subunits

“…The structural and functional properties of DWORF are unique among the regulins. Structurally, DWORF consists of a short, cytoplasmic helix (residues 1-13), a flexible linker centered around Pro 15 (residues 14-16), and a transmembrane helix (residues 17-35) (Figure 4A) [25,71]. Overall, the structural features of DWORF (Figure 4A) are similar to PLN (Figure 2A), though DWORF is a much shorter peptide (35 residues versus 52 residues, respectively).…”

Solid-state NMR study of structural heterogeneity of the apo WT mouse TSPO reconstituted in liposomes

“…Among the biomolecules of medical interest, membrane proteins such as GPCR suffer from spectral crowding induced by conformational plasticity [70] that, for most of them, limits their access for NMR investigation. High-magnetic field and MAS spinning above 60 kHz rotation frequency demonstrated that sensitivity and 1 H resolution can be significantly improved, notably for microcrystalline proteins available in small amounts [71] but also for membrane proteins [41]. Selective 13 C-isotope labelling schemes should alleviate the spectral crowding induced by the high percentage of hydrophobic amino acids present in -helical membrane proteins and therefore, they could help the detection of conformational plasticity of biologically systems by solid-state NMR.…”

“…Previous data on GB1 and Vpu recorded at different temperatures have shown their importance for the optimization of NMR sensitivity [37,38]. Most of the ssNMR data acquired on membrane proteins in lipids are based on CP rather than the INEPT element [39][40][41] since dipolar coupling transfer (through space) is expected to be more efficient than scalar (through bond) one. Scalar-based methods such as INEPT and notably INADEQUATE have been shown to work well for microcrystalline super-oxide dismutase under ultrafast magic-angle spinning [42].…”

Solid-State NMR: Methods for Biological Solids

“…The first approach involves the uniaxial alignment of protein molecules in lipid bilayers either mechanically on glass plates or with lipid bicelles that spontaneously align when placed in a magnetic field (section ). , This approach is primarily useful for investigating the structure, dynamics, and orientation of different transmembrane helices or membrane-bound segments of proteins and their complexes. , Magic angle spinning (MAS) is the second approach in ssNMR for characterizing the structure of biomolecules and has become an essential tool due to major developments in the last two decades. MAS approach entails mechanical spinning of biomolecular samples in rotors (cylindrical sample holders with turbines that spin using air) about an axis inclined at an angle of (∼54.7356°, the eponymous magic angle) with respect to the direction of the static magnetic field (section ).…”