Heteronuclear decoupling with rotor-synchronized phase-alternated cycles.

A new heteronuclear decoupling pulse sequence is introduced, dubbed ROtor-Synchronized Phase-Alternated Cycles (ROSPAC). It is based on a partial refocusing of the coherences (spin operator products or cross-terms) [Filip et al., J. Mag. Reson. 176, 2 (2005)] responsible for transverse spin-polarization dephasing, on the irradiation of a large pattern of radio-frequencies, and on a significant minimization of the cross-effects implying 1H chemical-shift anisotropy. Decoupling efficiency is analyzed by numerical simulations and experiments and compared to that of established decoupling sequences [swept-frequency two-pulse phase-modulated (TPPM), TPPM, small phase incremental alternation (SPINAL), refocused Continuous-wave (CWApa), and Rotor-Synchronized Hahn-Echo pulse train (RS-HEPT)]. It was found that ROSPAC offers good 1H offset robustness for a large range of chemical shifts and low radio-frequency (RF) powers, and performs very well in the ultra-fast magic-angle spinning (MAS) regime, where it is almost independent from RF power and permits it to avoid rotary-resonance recoupling conditions (v1 = nvr, n = 1, 2). It has the advantage that only the pulse lengths require optimization and has a low duty cycle in the pulsed decoupling regime. The efficiency of the decoupling sequence is demonstrated on a model microcrystalline sample of the model protein domain GB1 at 100 kHz MAS at 18.8 T.

Production and Preparation of Isotopically Labeled Human Membrane Proteins in Pichia pastoris for Fast-MAS-NMR Analyses.

Membrane proteins (MPs) comprise about one-third of the human proteome, playing critical roles in many physiological processes and associated disorders. Consistently, they represent one of the largest classes of targets for the pharmaceutical industry. Their study at the molecular level is however particularly challenging, resulting in a severe lack of structural and dynamic information that is hindering their detailed functional characterization and the identification of novel potent drug candidates.Magic Angle Spinning (MAS) NMR is a reliable and efficient method for the determination of protein structures and dynamics and for the identification of ligand binding sites and equilibria. MAS-NMR is particularly well suited for MPs since they can be directly analysed in a native-like lipid bilayer environment but used to require aggravating large amounts of isotope enriched material. The frequent toxicity of human MP overexpression in bacterial cultures poses an additional hurdle, resulting in the need for alternative (and often more costly) expression systems. The recent development of very fast (up to 150 kHz) MAS probes has revolutionized the field of biomolecular solid-state NMR enabling higher spectral resolution with significant reduction of the required sample, rendering eukaryotic expression systems cost-effective.Here is presented a set of accessible procedures validated for the production and preparation of eukaryotic MPs for Fast-MAS 1H-detected NMR analysis. The methodology is illustrated with the human copper uptake protein hCTR1 recombinantly produced and 13C-15N uniformly labeled with the versatile and affordable Pichia pastoris system. Subsequent purification procedures allow the recovery of mg amounts that are then reconstituted into liposome formulations compatible with solid-state NMR handling and analysis.

1H-Detected Biomolecular NMR under Fast Magic-Angle Spinning.

Since the first pioneering studies on small deuterated peptides dating more than 20 years ago, 1H detection has evolved into the most efficient approach for investigation of biomolecular structure, dynamics, and interactions by solid-state NMR. The development of faster and faster magic-angle spinning (MAS) rates (up to 150 kHz today) at ultrahigh magnetic fields has triggered a real revolution in the field. This new spinning regime reduces the 1H-1H dipolar couplings, so that a direct detection of 1H signals, for long impossible without proton dilution, has become possible at high resolution. The switch from the traditional MAS NMR approaches with 13C and 15N detection to 1H boosts the signal by more than an order of magnitude, accelerating the site-specific analysis and opening the way to more complex immobilized biological systems of higher molecular weight and available in limited amounts. This paper reviews the concepts underlying this recent leap forward in sensitivity and resolution, presents a detailed description of the experimental aspects of acquisition of multidimensional correlation spectra with fast MAS, and summarizes the most successful strategies for the assignment of the resonances and for the elucidation of protein structure and conformational dynamics. It finally outlines the many examples where 1H-detected MAS NMR has contributed to the detailed characterization of a variety of crystalline and noncrystalline biomolecular targets involved in biological processes ranging from catalysis through drug binding, viral infectivity, amyloid fibril formation, to transport across lipid membranes.

Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation.

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

Backbone assignment of crystalline E. coli maltose binding protein.

The E.coli maltose binding protein (MBP) is a 42.5 kDa molecule widely employed in many biotechnology applications. Because of its molecular size, it has become the main model system for the development of solution NMR methods adapted to large biomolecular targets. Here, we report virtually complete (~ 90%) backbone resonance assignments obtained on a microcrystalline sample of MBP with 1H-detected solid-state NMR at fast (> 100 kHz) magic-angle spinning. We additionally present the detailed description of the methodology employed for the preparation of the sample and the acquisition and analysis of the NMR spectra. The chemical shifts, obtained with a single uniformly 15N, 13C-labelled and fully-protonated sample and about 2 weeks on a 800 MHz NMR spectrometer, have been deposited to the BMRB under the accession number 50089.

Structural Analysis of an Antigen Chemically Coupled on Virus-Like Particles in Vaccine Formulation.

Structure determination of adjuvant-coupled antigens is essential for rational vaccine development but has so far been hampered by the relatively low antigen content in vaccine formulations and by their heterogeneous composition. Here we show that magic-angle spinning (MAS) solid-state NMR can be used to assess the structure of the influenza virus hemagglutinin stalk long alpha helix antigen, both in its free, unformulated form and once chemically coupled to the surface of large virus-like particles (VLPs). The sensitivity boost provided by high-field dynamic nuclear polarization (DNP) and proton detection at fast MAS rates allows to overcome the penalty associated with the antigen dilution. Comparison of the MAS NMR fingerprints between the free and VLP-coupled forms of the antigen provides structural evidence of the conservation of its native fold upon bioconjugation. This work demonstrates that high-sensitivity MAS NMR is ripe to play a major role in vaccine design, formulation studies, and manufacturing process development.

Multimodal Response to Copper Binding in Superoxide Dismutase Dynamics.

Copper/zinc superoxide dismutase (SOD) is a homodimeric metalloenzyme that has been extensively studied as a benchmark for structure-function relationships in proteins, in particular because of its implication in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis. Here, we investigate microcrystalline preparations of two differently metalated forms of SOD, namely, the fully mature functional Cu,Zn state and the E,Zn-SOD state in which the Cu site is empty. By using solid-state NMR with fast magic-angle spinning (MAS) at high magnetic fields (1H Larmor frequency of 800-1000 MHz), we quantify motions spanning a dynamic range from ns to ms. We determine that metal ion uptake does not act as a rigidification element but as a switch redistributing motional processes on different time scales, with coupling of the dynamics of histidine side chains and those of remote key backbone elements of the protein.

A ß-barrel for oil transport through lipid membranes: Dynamic NMR structures of AlkL.

The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops.

Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton-detected solid-state NMR.

Most commonly small outer membrane proteins, possessing between 8 and 12 ß-strands, are not involved in transport but fulfill diverse functions such as cell adhesion or binding of ligands. An intriguing exception are the 8-stranded ß-barrel proteins of the OmpW family, which are implicated in the transport of small molecules. A representative example is AlkL from Pseudomonas putida GPoI, which functions as a passive importer of hydrophobic molecules. This role is of high interest with respect to both fundamental biological understanding and industrial applications in biocatalysis, since this protein is frequently utilized in biotransformation of alkanes. While the transport function of AlkL is generally accepted, a controversy in the transport mechanism still exists. In order to address this, we are pursuing a structural study of recombinantly produced AlkL reconstituted in lipid bilayers using solid-state NMR spectroscopy. In this manuscript we present 1H, 13C and 15N chemical shift assignments obtained via a suite of 3D experiments employing high magnetic fields (1 GHz and 800 MHz) and the latest magic-angle spinning (MAS) approaches at fast (60-111) kHz rates. We additionally analyze the secondary structure prediction in comparison with those of published structures of homologous proteins.

Automated Backbone NMR Resonance Assignment of Large Proteins Using Redundant Linking from a Single Simultaneous Acquisition.

Thanks to magic-angle spinning (MAS) probes with frequencies of 60-100 kHz, the benefit of high-sensitivity 1H detection can now be broadly realized in biomolecular solid-state NMR for the analysis of microcrystalline, sedimented, or lipid-embedded preparations. Nonetheless, performing the assignment of all resonances remains a rate-limiting step in protein structural studies, and even the latest optimized protocols fail to perform this step when the protein size exceeds ∼20 kDa. Here, we leverage the benefits of fast (100 kHz) MAS and high (800 MHz) magnetic fields to design an approach that lifts this limitation. Through the creation, conservation, and acquisition of independent magnetization pathways within a single triple-resonance MAS NMR experiment, a single self-consistent data set can be acquired, providing enhanced sensitivity, reduced vulnerability to machine or sample instabilities, and highly redundant linking that supports fully automated peak picking and resonance assignment. The method, dubbed RAVASSA (redundant assignment via a single simultaneous acquisition), is demonstrated with the assignment of the largest protein to date in the solid state, the 42.5 kDa maltose binding protein, using a single fully protonated microcrystalline sample and 1 week of spectrometer time.

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.

Probing Membrane Protein Insertion into Lipid Bilayers by Solid-State NMR.

Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s-1 was determined for residues protected from exchange. Transfer within the protein, as tracked in the 15 N-1 H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s-1 for several resolved residues, explaining the site specificity.

Structural and functional characterization of the Curli adaptor protein CsgF.

Curli are functional amyloids that form a major part of the biofilm produced by many enterobacteriaceae. A multiprotein system around the outer membrane protein CsgG is in charge of the export and controlled propagation of the main Curli subunits, CsgA and CsgB. CsgF is essential for the linkage of the main amyloid-forming proteins to the cell surface. Here, we present a profound biochemical and biophysical characterization of recombinant CsgF, highlighted by a solution NMR structure of CsgF in the presence of dihexanoylphosphocholine micelles. Interestingly, CsgF contains large unstructured domains and does not show a globular fold. The data presented shed new light on the molecular mechanism of Curli amyloid surface attachment.

1H magic-angle spinning NMR evolves as a powerful new tool for membrane proteins.

Building on a decade of continuous advances of the community, the recent development of very fast (60 kHz and above) magic-angle spinning (MAS) probes has revolutionised the field of solid-state NMR. This new spinning regime reduces the 1H-1H dipolar couplings, so that direct detection of the larger magnetic moment available from 1H is now possible at high resolution, not only in deuterated molecules but also in fully-protonated substrates. Such capabilities allow rapid "fingerprinting" of samples with a ten-fold reduction of the required sample amounts with respect to conventional approaches, and permit extensive, robust and expeditious assignment of small-to-medium sized proteins (up to ca. 300 residues), and the determination of inter-nuclear proximities, relative orientations of secondary structural elements, protein-cofactor interactions, local and global dynamics. Fast MAS and 1H detection techniques have nowadays been shown to be applicable to membrane-bound systems. This paper reviews the strategies underlying this recent leap forward in sensitivity and resolution, describing its potential for the detailed characterization of membrane proteins.

Selective 1H-1H Distance Restraints in Fully Protonated Proteins by Very Fast Magic-Angle Spinning Solid-State NMR.

Very fast magic-angle spinning (MAS > 80 kHz) NMR combined with high-field magnets has enabled the acquisition of proton-detected spectra in fully protonated solid samples with sufficient resolution and sensitivity. One of the primary challenges in structure determination of protein is observing long-range 1H-1H contacts. Here we use band-selective spin-lock pulses to obtain selective 1H-1H contacts (e.g., HN-HN) on the order of 5-6 Å in fully protonated proteins at 111 kHz MAS. This approach is a major advancement in structural characterization of proteins given that magnetization can be selectively transferred between protons that are 5-6 Å apart despite the presence of other protons at shorter distance. The observed contacts are similar to those previously observed only in perdeuterated proteins with selective protonation. Simulations and experiments show the proposed method has performance that is superior to that of the currently used methods. The method is demonstrated on GB1 and a ß-barrel membrane protein, AlkL.

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.

Segmental Isotope Labeling of Insoluble Proteins for Solid-State NMR by Protein Trans-Splicing.

Solid-state NMR spectroscopy (ssNMR) is uniquely suited for atomic-resolution structural investigations of large protein assemblies, which are notoriously difficult to study due to their insoluble and non-crystalline nature. However, assignment ambiguities because of limited resolution and spectral crowding are currently major hurdles that quickly increase with the length of the polypeptide chain. The line widths of ssNMR signals are independent of proteins size, making segmental isotope labeling a powerful approach to overcome this limitation. It allows a scalable reduction of signal overlap, aids the assignment of repetitive amino acid sequences, and can be easily combined with other selective isotope labeling strategies. Here we present a detailed protocol for segmental isotope labeling of insoluble proteins using protein trans-splicing. Our protocol exploits the ability of many insoluble proteins, such as amyloid fibrils, to fold correctly under in vitro conditions. In combination with the robust trans-splicing efficiency of the intein DnaE from Nostoc punctiforme, this allows for high yields of segmentally labeled protein required for ssNMR analysis.

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.

Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy.

Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low-temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin-T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long-range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT.

Untangling a Repetitive Amyloid Sequence: Correlating Biofilm-Derived and Segmentally Labeled Curli Fimbriae by Solid-State NMR Spectroscopy.

Curli are functional bacterial amyloids produced by an intricate biogenesis machinery. Insights into their folding and regulation can advance our understanding of amyloidogenesis. However, gaining detailed structural information of amyloids, and their tendency for structural polymorphisms, remains challenging. Herein we compare high-quality solid-state NMR spectra from biofilm-derived and recombinantly produced curli and provide evidence that they adopt a similar, well-defined ß-solenoid arrangement. Curli subunits consist of five sequence repeats, resulting in severe spectral overlap. Using segmental isotope labeling, we obtained the unambiguous sequence-specific resonance assignments and secondary structure of one repeat, and demonstrate that all repeats are most likely structurally equivalent.