Protein Resonance Assignment Research Articles

Optimization of 15N–13C double-resonance NMR experiments under low temperature magic angle spinning dynamic nuclear polarization conditions

Dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) solid-state NMR carried out at 25 K enables rapid acquisition of multi-dimensional 13C–15N correlation spectra for protein structure studies and resonance assignment. Under commonly used DNP conditions, solvent deuteration reduces 1H–15N cross polarization (CP) efficiencies, necessitates more careful optimization, and requires longer high-power 15N radio-frequency pulses. The sensitivity of 2D heteronuclear correlation experiments is potentially impaired. Here we show that 2D 15N-13C experiments based on 13C-15N transferred echo double resonance (TEDOR) methods outperform 2D experiments based on CP transfers in a fully deuterated solvent, and are competitive with CP-based experiments when the solvent is only partially deuterated. Additionally, we show that optimization of TEDOR-based 2D experiments is simpler than optimization of CP-based experiments under 25 K MAS conditions.

Methyl probes in proteins for determining ligand binding mode in weak protein–ligand complexes

Structures of protein–ligand complexes provide critical information for drug design. Most protein–ligand complex structures are determined using X-ray crystallography, but where crystallography is not able to generate a structure for a complex, NMR is often the best alternative. However, the available tools to enable rapid and robust structure determination of protein–ligand complexes by NMR are currently limited. This leads to situations where projects are either discontinued or pursued without structural data, rendering the task more difficult. We previously reported the NMR Molecular Replacement (NMR2) approach that allows the structure of a protein–ligand complex to be determined without requiring the cumbersome task of protein resonance assignment. Herein, we describe the NMR2 approach to determine the binding pose of a small molecule in a weak protein–ligand complex by collecting sparse protein methyl-to-ligand NOEs from a selectively labeled protein sample and an unlabeled ligand. In the selective labeling scheme all methyl containing residues of the protein are protonated in an otherwise deuterated background. This allows measurement of intermolecular NOEs with greater sensitivity using standard NOESY pulse sequences instead of isotope-filtered NMR experiments. This labelling approach is well suited to the NMR2 approach and extends its utility to include larger protein–ligand complexes.

Protein resonance assignment by solid-state NMR based on 1H-detected 13C double-quantum spectroscopy at fast MAS.

Solid-state NMR spectroscopy is a powerful technique to study insoluble and non-crystalline proteins and protein complexes at atomic resolution. The development of proton (1H) detection at fast magic-angle spinning (MAS) has considerably increased the analytical capabilities of the technique, enabling the acquisition of 1H-detected fingerprint experiments in few hours. Here an approach based on double-quantum (DQ) 13C spectroscopy, detected on 1H, is proposed for fast MAS regime (> 60kHz) to perform the sequential assignment of insoluble proteins of small size, without any specific deuteration requirement. By combining two three-dimensional 1H detected experiments correlating a 13C DQ dimension respectively to its intra-residue and sequential 15N-1H pairs, a sequential walk through DQ (Ca + CO) resonance is obtained. The approach takes advantage of fast MAS to achieve an efficient sensitivity and the addition of a DQ dimension provides spectral features useful for the resonance assignment process.

Resonance assignment and secondary structure of DbpA protein from the European species, Borrelia afzelii

Decorin binding proteins (Dbps) mediate attachment of spirochetes in host organisms during the early stages of Lyme disease infection. Previously, different binding mechanisms of Dbps to glycosaminoglycans have been elucidated for the pathogenic species Borrelia burgdorferi sensu stricto and B. afzelii. We are investigating various European Borrelia spirochetes and their interactions at the atomic level using NMR. We report preparative scale recombinant expression of uniformly stable isotope enriched B. afzelii DbpA in Escherichia coli, its chromatographic purification, and solution NMR assignments of its backbone and sidechain 1H, 13C, and 15N atoms. This data was used to predict secondary structure propensity, which we compared to the North American B. burgdorferi sensu stricto and European B. garinii DbpA for which solution NMR structures had been determined previously. Backbone dynamics of DbpA from B. afzelii were elucidated from spin relaxation and heteronuclear NOE experiments. NMR-based secondary structure analysis together with the backbone dynamics characterization provided a first look into structural differences of B. afzelii DbpA compared to the North American species and will serve as the basis for further investigation of how these changes affect interactions with host components.

NMR backbone resonance assignment of Japanese encephalitis virus capsid protein.

Japanese encephalitis virus (JEV) is a flavivirus in the same family as West Nile virus (WNV), dengue virus (DENV) and yellow fever virus (YFV), which are transmitted by mosquitoes. About 68 thousand people are infected with JEV every year. In many Asian countries, JEV is the main cause of viral encephalitis. There are no specific antiviral drugs for Japanese encephalitis. Capsid protein C is the core protein of virus particles. Many studies have revealed that capsid protein C plays an important role in the life cycle of flaviviruses. Although the structure of JEV capsid protein (JEVC) has been determined by X-ray crystallography, the mechanism of how it assembles into an inner core to encapsulate the virus genome remains elusive. What's more, the disordered N-terminal region that is reported to affect its assembly is absent in the crystal structure. NMR spectroscopy has distinct advantages over other technologies in the characterization of conformational dynamics. Here we report the backbone 1H, 13C and 15N chemical shift assignments of JEVC by heteronuclear multidimensional spectroscopy and predict its secondary structure in solution using TALOS+.

Identification of a Potential Inhibitor of the FIV p24 Capsid Protein and Characterization of Its Binding Site.

Feline immunodeficiency virus (FIV) is a veterinary infective agent for which there is currently no efficient drug available. Drugs targeting the lentivirus capsid are currently under development for the treatment of human immunodeficiency virus 1 (HIV-1). Here we describe a lead compound that interacts with the FIV capsid. This compound, 696, modulates the in vitro assembly of and stabilizes the assembled capsid protein. To decipher the mechanism of binding of this compound to the protein, we performed the first nuclear magnetic resonance (NMR) assignment of the FIV p24 capsid protein. Experimental NMR chemical shift perturbations (CSPs) observed after the addition of 696 enabled the characterization of a specific binding site for 696 on p24. This site was further analyzed by molecular modeling of the protein:compound interaction, demonstrating a strong similarity with the binding sites of existing drugs targeting the HIV-1 capsid protein. Taken together, we characterized a promising capsid-interacting compound with a low cost of synthesis, for which derivatives could lead to the development of efficient treatments for FIV infection. More generally, our strategy combining the NMR assignment of FIV p24 with NMR CSPs and molecular modeling will be useful for the analysis of future compounds targeting p24 in the quest to identify an efficient treatment for FIV.

Four-dimensional NOE-NOE spectroscopy of SARS-CoV-2 Main Protease to facilitate resonance assignment and structural analysis.

Resonance assignment and structural studies of larger proteins by nuclear magnetic resonance (NMR) can be challenging when exchange broadening, multiple stable conformations, and H back-exchange of the fully deuterated chain pose problems. These difficulties arise for the SARS-CoV-2 Main Protease, a homodimer of 2 306 residues. We demonstrate that the combination of four-dimensional (4D) TROSY-NOESY-TROSY spectroscopy and 4D NOESY-NOESY-TROSY spectroscopy provides an effective tool for delineating the H-H dipolar relaxation network. In combination with detailed structural information obtained from prior X-ray crystallography work, such data are particularly useful for extending and validating resonance assignments as well as for probing structural features.

NMR Structure Determinations of Small Proteins Using only One Fractionally 20% 13C- and Uniformly 100% 15N-Labeled Sample.

Uniformly 13C- and 15N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of 13C-labeled glucose by a factor of five using a fractional 20% 13C- and 100% 15N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [13C, 15N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional 13C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% 13C, 100% 15N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% 15N-labeled and uniformly 100% [13C, 15N]-labeled samples of proteins. We determined the NMR structures of a small alpha-helical protein, the C domain of IgG-binding protein A from Staphylococcus aureus (SpaC), and a small beta-sheet protein, CBM64 module using [20% 13C, 100% 15N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [13C, 15N]-labeled sample. Our results suggest that one [20% 13C, 100% 15N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [13C, 15N]-labeling for backbone resonance assignments, NMR structure determination, 15N-relaxation analysis, and ligand–protein interaction.

Rapid nuclear magnetic resonance data acquisition with improved resolution and sensitivity for high-throughput metabolomic analysis.

Nuclear magnetic resonance (NMR)-based metabolomics has witnessed rapid advancements in recent years with the continuous development of new methods to enhance the sensitivity, resolution, and speed of data acquisition. Some of the approaches were earlier used for peptide and protein resonance assignments and have now been adapted to metabolomics. At the same time, new NMR methods involving novel data acquisition techniques, suited particularly for high-throughput analysis in metabolomics, have been developed. In this review, we focus on the different sampling strategies or data acquisition methods that have been developed in our laboratory and other groups to acquire NMR spectra rapidly with high sensitivity and resolution for metabolomics. In particular, we focus on the use of multiple receivers, phase modulation NMR spectroscopy, and fast-pulsing methods for identification and assignments of metabolites.

Applications of WaterControl to TOCSY and COSY experiments.

WaterControl is a solvent suppression method based on WATERGATE and PGSTEand is very efficient in selectively reducing the solvent signal in 1D pulse-acquire and 2D NOESY of protein solutions. In this study, the WaterControl technique was appended to two common 2D NMR methods used in resonance assignment of proteins, namely TOCSY and CLIP-COSY. Similar to that observed in regular 1D pulse-acquire and 2D NOESY, the incorporation of WaterControl in these 2D methods led to excellent solvent suppression superior to that obtained using W3- or W5-based WATERGATE sequences. The water signal was essentially eliminated in the TOCSY and CLIP-COSY with WaterControl while useful cross peaks around the water resonance at ω2 were preserved. This is in contrast to the 2D spectra obtained from the corresponding WATERGATE containing sequences, where these cross peaks in the ω2 region are usually suppressed together with the water resonance. These new WaterControl sequences provide significantly improved water suppression thereby facilitating protein NMR studies.

Detergent Titration as an Efficient Method for NMR Resonance Assignments of Membrane Proteins in Lipid-Bilayer Nanodiscs.

Lipid bilayer nanodiscs are an attractive tool to study membrane proteins in a detergent-free lipid-bilayer environment. In the case of NMR studies, a sequence-specific resonance assignment is required in order to gain structural and functional insights with atomic resolution. Although NMR backbone assignments of membrane proteins in detergents are available, they are largely absent for membrane proteins in nanodiscs due to unfavorable relaxation properties of the slowly tumbling membrane protein-nanodisc complex. The necessary residue-specific reassignment of resonances in nanodiscs is therefore extremely time and sample consuming and represents the fundamental bottleneck in the application of nanodiscs for NMR studies. Here we present an elegant and fast solution to the problem. We show that a resonance assignment in detergent micelles can be transferred to a spectrum recorded in nanodiscs via detergent titration. The procedure requires that lipid-detergent exchange kinetics are in the fast exchange regime in order to follow linear and nonlinear peak shift trajectories with increasing detergent concentration. We demonstrate the feasibility of the approach on the 148-residue membrane protein OmpX. The titration method is then applied to VDAC, a 19-stranded β-barrel with 283 residues, for which 67% of the detergent assignment could be transferred to the nanodisc spectrum. We furthermore show that this method also works for the largest currently assigned membrane protein, BamA with 398 residues. The method is applicable for backbone amide and side chain methyl groups and represents a time and cost-effective assignment method, for example, to investigate membrane protein allostery and drug binding in a more natural and detergent-free lipid bilayer.

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 resonance assignment by BSH-CP-based 3D solid-state NMR experiments: A practical guide.

Solid-state NMR (ssNMR) spectroscopy has evolved into a powerful method to obtain structural information and to study the dynamics of proteins at atomic resolution and under physiological conditions. The method is especially well suited to investigate insoluble and noncrystalline proteins that cannot be investigated easily by X-ray crystallography or solution NMR. To allow for detailed analysis of ssNMR data, the assignment of resonances to the protein atoms is essential. For this purpose, a set of three-dimensional (3D) spectra needs to be acquired. Band-selective homo-nuclear cross-polarization (BSH-CP) is an effective method for magnetization transfer between carbonyl carbon (CO) and alpha carbon (CA) atoms, which is an important transfer step in multidimensional ssNMR experiments. This tutorial describes the detailed procedure for the chemical shift assignment of the backbone atoms of 13 C-15 N-labeled proteins by BSH-CP-based 13 C-detected ssNMR experiments. A set of six 3D experiments is used for unambiguous assignment of the protein backbone as well as certain side-chain resonances. The tutorial especially addresses scientists with little experience in the field of ssNMR and provides all the necessary information for protein assignment in an efficient, time-saving approach.

Automatic structure-based NMR methyl resonance assignment in large proteins

Isotopically labeled methyl groups provide NMR probes in large, otherwise deuterated proteins. However, the resonance assignment constitutes a bottleneck for broader applicability of methyl-based NMR. Here, we present the automated MethylFLYA method for the assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA is applied to five proteins (28–358 kDa) comprising a total of 708 isotope-labeled methyl groups, of which 612 contribute NOESY cross peaks. MethylFLYA confidently assigns 488 methyl groups, i.e. 80% of those with NOESY data. Of these, 459 agree with the reference, 6 were different, and 23 were without reference assignment. MethylFLYA assigns significantly more methyl groups than alternative algorithms, has an average error rate of 1%, modest runtimes of 0.4–1.2 h, and can handle arbitrary isotope labeling patterns and data from other types of NMR spectra.

BaMORC: A Software Package for Accurate and Robust 13C Reference Correction of Protein NMR Spectra.

We describe BaMORC, a software package that performs 13C chemical shifts reference correction for either assigned or unassigned peak lists derived from protein NMR spectra. BaMORC provides an intuitive command line interface that allows non-NMR experts to detect and correct 13C chemical shift referencing errors of unassigned peak lists at the very beginning of NMR data analysis, further lowering the bar of expertise required for effective protein NMR analysis. Furthermore, BaMORC provides an application programming interface for integration into sophisticated protein NMR data analysis pipelines, both before and after the protein resonance assignment step.

Experiments with direct detection of multiple FIDs.

Pulse schemes with direct observation of multiple free induction decays (FIDs) offer a dramatic increase in the spectral information content of NMR experiments and often yield substantial improvement in measurement sensitivity per unit time. Availability of multiple receivers on the state-of-the-art commercial spectrometers allows spectra from different nuclear species to be recorded in parallel routinely. Experiments with multi-FID detection have been designed with both, homonuclear and multinuclear acquisition. We provide a brief overview of such techniques designed for applications in liquid- and solid- state NMR as well as in hyperpolarized samples. Here we show how these techniques have led to design of experiments that allow structure elucidation of small molecules and resonance assignment in proteins from a single measurement. Probes with multiple RF micro-coils routed to multiple NMR receivers provide an alternative way of increasing the throughput of modern NMR systems. Solid-state NMR experiments have also benefited immensely from both parallel and simultaneous FID acquisition in a variety of multi-dimensional pulse schemes. We believe that multi-FID detection will become an essential component of the future NMR methodologies effectively increasing the information content of NMR experiments and reducing the cost of NMR analysis.

15N, 13C and 1H resonance assignments of FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus

Invasive fungal infections are a leading cause of death in immunocompromised patients and remain difficult to treat since fungal pathogens, like mammals, are eukaryotes and share many orthologous proteins. As a result, current antifungal drugs have limited clinical value, are sometimes toxic, can adversely affect human reaction pathways and are increasingly ineffective due to emerging resistance. One potential antifungal drug, FK506, establishes a ternary complex between the phosphatase, calcineurin, and the 12-kDa peptidyl-prolyl isomerase FK506-binding protein, FKBP12. It has been well established that calcineurin, highly conserved from yeast to mammals, is necessary for invasive fungal disease and is inhibited when in complex with FK506/FKBP12. Unfortunately, FK506 is also immunosuppressive in humans, precluding its usage as an antifungal drug, especially in immunocompromised patients. Whereas the homology between human and fungal calcineurin proteins is > 80%, the human and fungal FKBP12s share 48–58% sequence identity, making them more amenable candidates for drug targeting efforts. Here we report the backbone and sidechain NMR assignments of recombinant FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus in the apo form and compare these to the backbone assignments of the FK506 bound form. In addition, we report the backbone assignments of the apo and FK506 bound forms of the Homo sapiens FKBP12 protein for evaluation against the fungal forms. These data are the first steps towards defining, at a residue specific level, the impacts of FK506 binding to fungal and mammalian FKBP12 proteins. Our data highlight differences between the human and fungal FKBP12s that could lead to the design of more selective anti-fungal drugs.

Rapid NMR assignments of intrinsically disordered proteins using two-dimensional 13C-detection based experiments.

An approach for rapid backbone resonance assignments in proteins using only two 2D NMR experiments is presented. The new method involves a combination of high-resolution 13Cα-detected NMR experiments and selective unlabeling of amino acid residues. The 13C detected 2D hNCA and 2D hNcoCA spectra of a uniformly labeled sample of the protein are analysed in concert with the 2D hNCA spectrum obtained for a selectively unlabeled sample. The combinatorial set of amino acid residues for selective unlabeling is chosen optimally to maximize the assignments. The method is useful for rapid assignment of proteins with low stability such as intrinsically disordered proteins and is applicable to deuterated proteins. This approach helped in assignments of 14.5 kDa human α-synuclein during the course of its aggregation.

Nuclear magnetic resonance structure-based drug design.

Nuclear magnetic resonance structure-based drug design.

Efficient 15N-13C Polarization Transfer by Third-Spin-Assisted Pulsed Cross-Polarization Magic-Angle-Spinning NMR for Protein Structure Determination.

We introduce a pulsed third-spin-assisted recoupling experiment that produces high-intensity long-range 15N-13C cross peaks using low radiofrequency (rf) energy. This Proton-Enhanced Rotor-echo Short-Pulse IRradiATION Cross-Polarization (PERSPIRATIONCP) pulse sequence operates with the same principle as the Proton-Assisted Insensitive-Nuclei Cross-Polarization (PAINCP) experiment but uses only a fraction of the rf energy by replacing continuous-wave 13C and 15N irradiation with rotor-echo 90° pulses. Using formyl-Met-Leu-Phe (f-MLF) and β1 immunoglobulin binding domain of protein G (GB1) as model proteins, we demonstrate experimentally how PERSPIRATIONCP polarization transfer depends on the CP contact time, rf power, pulse flip angle, and 13C carrier frequency and compare the PERSPIRATIONCP performance with the performances of PAINCP, RESPIRATIONCP, and SPECIFICCP for measuring 15N-13C cross peaks. PERSPIRATIONCP achieves long-range 15N-13C transfer and yields higher cross peak-intensities than that of the other techniques. Numerical simulations reproduce the experimental trends and moreover indicate that PERSPIRATIONCP relies on 15N-1H and 13C-1H dipolar couplings rather than 15N-13C dipolar coupling for polarization transfer. Therefore, PERSPIRATIONCP is an rf-efficient and higher-sensitivity alternative to PAINCP for measuring long-range 15N-13C correlations, which are essential for protein resonance assignment and structure determination. Using cross peaks from two PERSPIRATIONCP 15N-13C correlation spectra as the sole distance restraints, supplemented with (φ, ψ) torsion angles obtained from chemical shifts, we calculated the GB1 structure and obtained a backbone root-mean-square deviation of 2.0 Å from the high-resolution structure of the protein. Therefore, this rf-efficient PERSPIRATIONCP method is useful for obtaining many long-range distance restraints for protein structure determination.