Andrea Bertarello

Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning

Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

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

Significance Protein structure determination is key to the detailed description of many biological processes. The critical factor that would allow general application of magic-angle spinning (MAS) solid-state NMR to this end is improvement in sensitivity and resolution for as many nuclear spins as possible. This is achieved here with detection of resolved 1H resonances in protonated proteins by increasing MAS rates to frequencies of 100 kHz and above. For large proteins and assemblies, ultrafast spinning narrows spectral resonances better than Brownian motion on which solution NMR relies, removing a fundamental barrier to the NMR study of large systems. This is exploited here to determine the de novo structure of a 28-kDa protein dimer in a 2.5-MDa viral capsid assembly. 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 1H-1H 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 Spectroscopic Assignment of Backbone and Side-Chain Protons in Fully Protonated Proteins: Microcrystals, Sedimented Assemblies, and Amyloid Fibrils.

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100-111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.

Dynamic Nuclear Polarization enhanced MAS NMR spectroscopy for structural analysis of HIV-1 protein assemblies

Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

Paramagnetic Properties of a Crystalline Iron–Sulfur Protein by Magic-Angle Spinning NMR Spectroscopy

We present the first solid-state NMR study of an iron-sulfur protein. The combined use of very fast (60 kHz) magic-angle spinning and tailored radiofrequency irradiation schemes allows the detection and the assignment of most of the 1H and 13C resonances of the oxidized high-potential iron-sulfur protein I from Ectothiorhodospira halophila (EhHiPIP I), including those in residues coordinating the Fe4S4 cluster. For these residues, contact shifts as large as 100 and 400 ppm for 1H and 13C resonances, respectively, were observed, which represent the most shifted solid-state NMR signals ever measured in metalloproteins. Interestingly, by targeting EhHiPIP I in a crystalline environment, we were able to capture distinct paramagnetic signatures from the two conformations present in the asymmetric unit. The magnetic properties of the system were verified by following the temperature dependence of the contact-shifted cysteine resonances.

Chapter 6:Solid-state NMR of Paramagnetic Proteins

In this chapter we will briefly summarize how the presence of paramagnetic centers affects the NMR properties of paramagnetic proteins in the solid state and describe how the experimental strategies used in solution should be modified to characterize these systems. In parallel, we will provide some case studies from the recent literature that highlight the tremendous potential of solid-state NMR for the characterization of paramagnetic proteins.