Protein NMR Resonance Assignment without Spectral Analysis: 5D SOlid-State Automated Projection SpectroscopY (SO-APSY).

Narrow proton signals, high sensitivity, and efficient coherence transfers provided by fast magic-angle spinning at high magnetic fields make automated projection spectroscopy feasible for the solid-state NMR analysis of proteins. We present the first ultrahigh dimensional implementation of this approach, where 5D peak lists are reconstructed from a number of 2D projections for protein samples of different molecular sizes and aggregation states, which show limited dispersion of chemical shifts or inhomogeneous broadenings. The resulting datasets are particularly suitable to automated analysis and yield rapid and unbiased assignments of backbone resonances.

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR.

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.

Dynamic Nuclear Polarization-Enhanced Biomolecular NMR Spectroscopy at High Magnetic Field with Fast Magic-Angle Spinning.

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.

Is protein deuteration beneficial for proton detected solid-state NMR at and above 100 kHz magic-angle spinning?

1H-detection in solid-state NMR of proteins has been traditionally combined with deuteration for both resolution and sensitivity reasons, with the optimal level of proton dilution being dependent on MAS rate. Here we present 1H-detected 15N and 13C CP-HSQC spectra on two microcrystalline samples acquired at 60 and 111 kHz MAS and at ultra-high field. We critically compare the benefits of three labeling schemes yielding different levels of proton content in terms of resolution, coherence lifetimes and feasibility of scalar-based 2D correlations under these experimental conditions. We observe unexpectedly high resolution and sensitivity of aromatic resonances in 2D 13C-1H correlation spectra of protonated samples. Ultrafast MAS reduces or even removes the necessity of 1H dilution for high-resolution 1H-detection in biomolecular solid-state NMR. It yields 15N,1H and 13C,1H fingerprint spectra of exceptional resolution for fully protonated samples, with notably superior 1H and 13C lineshapes for side-chain resonances.

Protein residue linking in a single spectrum for magic-angle spinning NMR assignment.

Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.

Deciphering the role of CD1e protein in mycobacterial phosphatidyl-myo-inositol mannosides (PIM) processing for presentation by CD1b to T lymphocytes.

Lipids are important antigens that induce T cell-mediated specific immune responses. They are presented to T lymphocytes by a specific class of MHC-I like proteins, termed CD1. The majority of the described CD1-presented mycobacterial antigens are presented by the CD1b isoform. We previously demonstrated that the stimulation of CD1b-restricted T cells by the hexamannosylated phosphatidyl-myo-inositol (PIM(6)), a family of mycobacterial antigens, requires a prior partial digestion of the antigen oligomannoside moiety by α-mannosidase and that CD1e is an accessory protein absolutely required for the generation of the lipid immunogenic form. Here, we show that CD1e behaves as a lipid transfer protein influencing lipid immunoediting and membrane transfer of PIM lipids. CD1e selectively assists the α-mannosidase-dependent digestion of PIM(6) species according to their degree of acylation. Moreover, CD1e transfers only diacylated PIM from donor to acceptor liposomes and also from membranes to CD1b. This study provides new insight into the molecular mechanisms by which CD1e contributes to lipid immunoediting and CD1-restricted presentation to T cells.

Deciphering sulfoglycolipids of Mycobacterium tuberculosis.

For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways.

Probing Protein Dynamics Using Multifield Variable Temperature NMR Relaxation and Molecular Dynamics Simulation.

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.

Lysosomal Lipases PLRP2 and LPLA2 Process Mycobacterial Multi-acylated Lipids and Generate T Cell Stimulatory Antigens.

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.