Project description:Magic angle spinning (MAS) is commonly used in nuclear magnetic resonance of solids to improve spectral resolution. Rather than using cylindrical rotors for MAS, we demonstrate that spherical rotors can be spun stably at the magic angle. Spherical rotors conserve valuable space in the probe head and simplify sample exchange and microwave coupling for dynamic nuclear polarization. In this current implementation of spherical rotors, a single gas stream provides bearing gas to reduce friction, drive propulsion to generate and maintain angular momentum, and variable temperature control for thermostating. Grooves are machined directly into zirconia spheres, thereby converting the rotor body into a robust turbine with high torque. We demonstrate that 9.5-mm-outside diameter spherical rotors can be spun at frequencies up to 4.6 kHz with N2(g) and 10.6 kHz with He(g). Angular stability of the spinning axis is demonstrated by observation of 79Br rotational echoes out to 10 ms from KBr packed within spherical rotors. Spinning frequency stability of ±1 Hz is achieved with resistive heating feedback control. A sample size of 36 μl can be accommodated in 9.5-mm-diameter spheres with a cylindrical hole machined along the spinning axis. We further show that spheres can be more extensively hollowed out to accommodate 161 μl of the sample, which provides superior signal-to-noise ratio compared to traditional 3.2-mm-diameter cylindrical rotors.
Project description:Viruses, relatively simple pathogens, are able to replicate in many living organisms and to adapt to various environments. Conventional atomic-resolution structural biology techniques, X-ray crystallography and solution NMR spectroscopy provided abundant information on the structures of individual proteins and nucleic acids comprising viruses; however, viral assemblies are not amenable to analysis by these techniques because of their large size, insolubility, and inherent lack of long-range order. In this article, we review the recent advances in magic angle spinning NMR spectroscopy that enabled atomic-resolution analysis of structure and dynamics of large viral systems and give examples of several exciting case studies.
Project description:The structure of the transmembrane domain of the pH-activated bacterial potassium channel KcsA has been extensively characterized, yet little information is available on the structure of its cytosolic, functionally critical N- and C-termini. This study presents high-resolution magic angle spinning (HR-MAS) and fractional deuteration as tools to study these poorly resolved regions for proteoliposome-embedded KcsA. Using 1H-detected HR-MAS NMR, we show that the C-terminus transitions from a rigid structure to a more dynamic structure as the solution is rendered acidic. We make previously unreported assignments of residues in the C-terminus of lipid-embedded channels. These data agree with functional models of the C-terminus-stabilizing KcsA tetramers at a neutral pH with decreased stabilization effects at acidic pH. We present evidence that a C-terminal truncation mutation has a destabilizing effect on the KcsA selectivity filter. Finally, we show evidence of hydrolysis of lipids in proteoliposome samples during typical experimental timeframes.
Project description:Techniques for atomic-resolution structural biology have evolved during the past several decades. Breakthroughs in instrumentation, sample preparation, and data analysis that occurred in the past decade have enabled characterization of viruses with an unprecedented level of detail. Here we review the recent advances in magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy for structural analysis of viruses and viral assemblies. MAS NMR is a powerful method that yields information on 3D structures and dynamics in a broad range of experimental conditions. After a brief introduction, we discuss recent structural and functional studies of several viruses investigated with atomic resolution at various levels of structural organization, from individual domains of a membrane protein reconstituted into lipid bilayers to virus-like particles and intact viruses. We present examples of the unique information revealed by MAS NMR about drug binding, conduction mechanisms, interactions with cellular host factors, and DNA packaging in biologically relevant environments that are inaccessible by other methods.
Project description:In this work, we demonstrate the first pulsed electron paramagnetic resonance (EPR) experiments performed under magic angle spinning (MAS) at high magnetic field. Unlike nuclear magnetic resonance (NMR) and dynamic nuclear polarization (DNP), commonly performed at high magnetic fields and under MAS to maximize sensitivity and resolution, EPR is usually measured at low magnetic fields and, with the exception of the Spiess group work in the late 1990s, never under MAS, due to great instrumentational challenges. This hampers the investigation of DNP mechanisms, in which electron spin dynamics play a central role, because no experimental data about the latter under DNP-characteristic conditions are available. We hereby present our dedicated, homebuilt MAS-EPR probehead and show the pulsed MAS-EPR spectra of P1 center diamond defect recorded at 7 tesla. Our results reveal unique effects of MAS on EPR line shape, intensity, and signal dephasing. Time-domain simulations reproduce the observed changes in the line shapes and the trends in the signal intensity.
Project description:G protein-coupled receptors (GPCRs) have a simple seven transmembrane helix architecture which has evolved to recognize a diverse number of chemical signals. The more than 800 GPCRs encoded in the human genome function as receptors for vision, smell and taste, and mediate key physiological processes. Consequently, these receptors are a major target for pharmaceuticals. Protein crystallography and electron cryo-microscopy have provided high resolution structures of many GPCRs in both active and inactive conformations. However, these structures have not sparked a surge in rational drug design, in part because GPCRs are inherently dynamic and the structural changes induced by ligand or drug binding to stabilize inactive or active conformations are often subtle rearrangements in packing or hydrogen-bonding interactions. NMR spectroscopy provides a sensitive probe of local structure and dynamics at specific sites within these receptors as well as global changes in receptor structure and dynamics. These methods can also capture intermediate states and conformations with low populations that provide insights into the activation pathways. We review the use of solid-state magic angle spinning NMR to address the structure and activation mechanisms of GPCRs. The focus is on the large and diverse class A family of receptors. We highlight three specific class A GPCRs in order to illustrate how solid-state, as well as solution-state, NMR spectroscopy can answer questions in the field involving how different GPCR classes and subfamilies are activated by their associated ligands, and how small molecule drugs can modulate GPCR activation.
Project description:DNP-enhanced solid-state NMR spectroscopy under magic angle spinning (MAS) is rapidly developing into a powerful analytical tool to investigate the structure of a wide range of solid materials, because it provides unsurpassed sensitivity gains. Most developments and applications of DNP MAS NMR were so far reported at moderate spinning frequencies (up to 14 kHz using 3.2 mm rotors). Here, using a 1.3 mm MAS DNP probe operating at 18.8 T and ∼100 K, we show that signal amplification factors can be increased by up to a factor two when using smaller volume rotors as compared to 3.2 mm rotors, and report enhancements of around 60 over a range of sample spinning rates from 10 to 40 kHz. Spinning at 40 kHz is also shown to increase (29)Si coherence lifetimes by a factor three as compared to 10 kHz, substantially increasing sensitivity in CPMG type experiments. The contribution of quenching effects to the overall sensitivity gain at very fast MAS is evaluated, and applications are reported on a functionalised mesostructured organic-inorganic material.
Project description:Protein torsion angles define the backbone secondary structure of proteins. Magic-angle spinning (MAS) NMR methods using carbon detection have been developed to measure torsion angles by determining the relative orientation between two anisotropic interactions─dipolar coupling or chemical shift anisotropy. Here we report a new proton-detection based method to determine the backbone torsion angle by recoupling NH and CH dipolar couplings within the HCANH pulse sequence, for protonated or partly deuterated samples. We demonstrate the efficiency and precision of the method with microcrystalline chicken α spectrin SH3 protein and the influenza A matrix 2 (M2) membrane protein, using 55 or 90 kHz MAS. For M2, pseudo-4D data detect a turn between transmembrane and amphipathic helices.
Project description:3D printing has evolved into an invaluable tool for rapid and cost-effective production of intricate parts. In this paper we describe 3D printing and other rapid prototyping methods to fabricate 3.2 mm stators and drive caps for use in magic angle spinning (MAS) NMR experiments. These components can be fabricated with the assistance of computer-aided design (CAD) software and at a fraction of the cost of commercial parts. Additionally, we show that the performance of these 3D printed stators and drive caps is comparable to commercially available systems and that they have significant advantages over their machined counterparts.
Project description:Here we report the structure of Opa60 in lipid bilayers using proton-detected magic-angle spinning nuclear magnetic resonance (MAS NMR). Preparations including near-native oligosaccharide lipids reveal a consistent picture of a stable transmembrane beta barrel with a minor increase in the structured region as compared with the previously reported detergent structure. The large variable loops known to interact with host proteins could not be detected, confirming their dynamic nature even in a lipid bilayer environment. The structure provides a starting point for investigation of the functional role of Opa60 in gonococcal infection, which is understood to involve interaction with host proteins. At the same time, it demonstrates the recent advances in proton-detected methodology for membrane protein structure determination at atomic resolution by MAS NMR.