Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of 1 H-1 H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.NMR spectroscopy | magic-angle spinning | protein structures | proton detection | viral nucleocapsids D espite tremendous progress in the analysis of biomolecular samples over the last two decades (1-7), routine application of magic-angle spinning (MAS) NMR in biology is still limited by the inherently low sensitivity. The direct detection of proton resonances is a straightforward way to counter this problem, but entails a trade-off with resolution due to the strong homonuclear dipolar interactions among proton nuclei. High-resolution proton-detected methods were first demonstrated with modest spinning frequencies by today's standards (∼10 kHz) and relied on a reduction of 1 H-1 H couplings by high levels of dilution with deuterium, typically perdeuteration, and complete (8, 9) or partial (10-12) protonation at exchangeable sites. The need for narrow proton resonances without such extreme levels of deuteration has motivated a continuous technological development, resulting in a dramatic increase in the available spinning frequency (13)(14)(15)(16)(17)(18)(19)(20).At MAS frequencies of 40-60 kHz, deuteration and 100% reprotonation at exchangeable sites, primarily amide protons, result in resolved and sensitive spectra, similar in quality to the case of higher dilution levels and lower spinning frequencies (21-23). This opens the way to rapid sequential assignment of backbone resonances (24-27), as well as to the unambiguous measurement of detailed structural and dynamical parameters (28-32). A further increase in the MAS frequency to 100 kHz allows resonance assignment (20), a structure determination of a model protein (16), and interaction studies (15) with as little as 0.5 mg of sample. However, a high deuteration level severely limits observation of side-chain s...

Dynamic nuclear polarization (DNP) is apowerful way to overcome the sensitivity limitation of magic-anglespinning (MAS) NMR experiments.However,the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures and in the presence of paramagnetic radicals.H igh-quality DNP-enhanced NMR spectra of the Acinetobacter phage 205 (AP205) nucleocapsid can be obtained by combining high magnetic field (800 MHz) and fast MAS (40 kHz). These conditions yield enhanced resolution and long coherence lifetimes allowing the acquisition of resolved 2D correlation spectra and of previously unfeasible scalar-based experiments.T his enables the assignment of aromatic resonances of the AP205 coat protein and its packaged RNA, as well as the detection of long-range contacts, which are not observed at room temperature,o pening new possibilities for structure determination.Dynamic nuclear polarization (DNP) allows transfer of polarization from the unpaired electrons of ap aramagnetic center to the surrounding nuclei, enhancing the magic-anglespinning (MAS) NMR signals by several orders of magnitude. [1] Thetechnique has been successfully applied in various fields from chemistry and materials to structural and cell biology.H owever,d espite convincing demonstrations on fibrils, [2] membrane-embedded proteins, [3] virus capsids, [4] and whole-cell assemblies, [5] thes ensitivity enhancement provided by DNP on biological systems is always associated with an umber of issues.W hile high-resolution solid-state MAS NMR spectra of proteins can be nowadays obtained at temperatures above 273 K, the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures (at about 100 K) and in the presence of paramagnetic radicals.F urthermore,D NP spectra of protein samples are currently almost exclusively acquired at moderate magnetic fields (usually 9.4 T) for maximum DNP enhancements and using 3.2 mm rotors with MAS rates up to 15 kHz. Under these experimental conditions,t he lines are broadened by both homogeneous broadening,o wing to incomplete averaging of nuclear and hyperfine interactions, and inhomogeneous broadening,a rising from protein conformational disorder. [6] Thelack of resolution associated with the DNP enhancements is therefore amajor obstacle for the detailed structural study of uniformly labeled biomolecular samples.This limitation has been previously addressed by using increased temperatures (180-200 K) [7] and higher magnetic field strengths [8] at the expense of lower DNP enhancements, as well as by exploring improved sample preparation methods. [9] Whilst these initial attempts have shown the potential for improving resolution in DNP spectra, af urther improvement is imperative to take full advantage of the signal amplification potentially provided by DNP.O ne factor that holds promise is the MAS rate.T he availability of prob...

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Dynamic nuclear polarization (DNP) is a powerful way to overcome the sensitivity limitation of magic-angle-spinning (MAS) NMR experiments. However, the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures and in the presence of paramagnetic radicals. High-quality DNP-enhanced NMR spectra of the Acinetobacter phage 205 (AP205) nucleocapsid can be obtained by combining high magnetic field (800 MHz) and fast MAS (40 kHz). These conditions yield enhanced resolution and long coherence lifetimes allowing the acquisition of resolved 2D correlation spectra and of previously unfeasible scalar-based experiments. This enables the assignment of aromatic resonances of the AP205 coat protein and its packaged RNA, as well as the detection of long-range contacts, which are not observed at room temperature, opening new possibilities for structure determination.

Understanding the interplay between protein function and dynamics is currently one of the fundamental challenges of physical biology. Recently, a method using variable temperature solid-state nuclear magnetic resonance relaxation measurements has been proposed for the simultaneous measurement of 12 different activation energies reporting on distinct dynamic modes in the protein GB1. Here, we extend this approach to measure relaxation at multiple magnetic field strengths, allowing us to better constrain the motional models and to simultaneously evaluate the robustness and physical basis of the method. The data reveal backbone and side-chain motions, exhibiting low-and high-energy modes with temperature coefficients around 5 and 25 kJ•mol −1. The results are compared to variable temperature molecular dynamics simulation of the crystal lattice, providing further support for the interpretation of the experimental data in terms of molecular motion.

Complex antigens require processing within antigen-presenting cells (APCs) to form T cell stimulatory complexes with CD1 antigen-presenting molecules. It remains unknown whether lipids with multi-acylated moieties also necessitate digestion by lipases to become capable of binding CD1 molecules and stimulate T cells. Here, we show that the mycobacterial tetra-acylated glycolipid antigens phosphatidyl-myo-inositol mannosides (PIM) are digested to di-acylated forms by pancreatic lipase-related protein 2 (PLRP2) and lysosomal phospholipase A2 (LPLA2) within APCs. Recombinant PLRP2 and LPLA2 removed the sn1- and sn2-bound fatty acids from the PIM glycerol moiety, as revealed by mass spectrometry and nuclear magnetic resonance studies. PLRP2 or LPLA2 gene silencing in APCs abolished PIM presentation to T cells, thus revealing an essential role of both lipases in vivo. These findings show that endosomal lipases participate in lipid antigen presentation by processing lipid antigens and have a role in T cell immunity against mycobacteria.

Background: To become antigenic, mycobacterial hexamannosylated phosphatidyl-myo-inositol (PIM 6 ) undergo CD1e-assisted ␣-mannosidase processing. Results: Measuring membrane-to-membrane PIM transfer, CD1e selectively transferred diacylated PIM species in accordance with the fact that liposome-inserted di-acylated PIM 6 were the only PIM species digested into antigenic molecules by ␣-mannosidase. Conclusion: CD1e assists PIM processing through its lipid transfer protein property. Significance: This study reveals the molecular mechanisms by which CD1e contributes to lipid immunoediting.

Narrow proton signals, high sensitivity, and efficient coherence transfers provided by fast magic-angle spinning at high magnetic fields make automated projection spectroscopy feasible in protein solid-state NMR. We present the first ultra-high dimensional implementation of this approach where 5D peak lists are reconstructed from a number of 2D projections for protein samples of different molecular size and aggregation state, featuring limited dispersion of chemical shifts or inhomogeneous broadenings. The resulting datasets are particularly suitable to automated analysis, yielding rapid and unbiased backbone resonance assignments. under the European Union's Horizon 2020 research and innovation programme (ERC-2015-CoG GA 648974), by the CNRS (IR-RMN FR3050), and by the EU-project iNext (GA 653706). HO was supported by the Westpac Foundation with a Future Leaders Scholarship, and JS by the EC's REA with a MSCA fellowship (GA 661799).