Sequential Resonance Assignments Research Articles

NMR side-chain assignments of the Crimean–Congo hemorrhagic fever virus glycoprotein n cytosolic domain

Abstract. We assigned the side-chain resonances of the Crimean–Congo hemorrhagic fever virus (CCHFV) glycoprotein n (Gn) cytosolic domain that is 69 amino acids long to complete the backbone resonances previously published by Estrada et al. (2011). The process was facilitated by three factors. First, sample preparation using cell-free protein synthesis (CFPS) was completed in less than 2 d and allowed for correct zinc finger formation by adding zinc ions to the reaction. Second, access to NMR platforms with standardized pulse sequences allowed for data acquisition in 18 d. Third, data analysis using the online platform NMRtist allowed sequential resonance assignments to be made in a day, and assignments were verified and finalized in less than a week. Our work thus provides an example of how NMR assignments, including side chains, of small and well-behaved proteins can be approached in a rapid routine, at protein concentrations of 150 µM.

1Hβ chemical shift-based phase modulated NMR methods for fast identification of amino acid types in proteins

Here we report two phase modulated NMR experiments: PM-2D HN(CACBHB) and PM-2D HN(HB), that use 1Hβ chemical shifts to rapidly identify amino acid type in proteins. The magnetization on the 1Hβ spins during the experiments is allowed to evolve for a fixed evolution period that results in phase modulation (positive or negative) of the cross peaks corresponding to various amino acid residues on their 2D HN projections, resembling a typical 2D [1H–15N]-HSQC spectrum. All amino acids except glycine can be categorized into three discernible groups based on their 1Hβ chemical shifts, resulting in unique phase patterns at different fixed evolution periods for 1Hβ, thus facilitating their identification. Remarkably, the PM-2D HN(HB) stands out among all amino acid type identification NMR techniques for its applicability with cost-effective and most routinely employed 15N-labeled protein samples for NMR studies. Furthermore, when combined effectively with the 13Cβ chemical shift-based phase modulated NMR method (PM-2D HN(CACB)), these methods resolved the identification of large groups of amino acids into relatively smaller groups. Moreover, these techniques can accelerate the sequence-specific sequential resonance assignment (SSRA) process and would help in fast tracking of assigned NMR signals exhibiting chemical shift perturbation on the 2D [1H–15N]-HSQC spectrum of proteins during various experiments (e.g., temperature change, pH change, and protein or ligand or cofactor binding) as well as in site-directed mutagenesis.

Solution-state NMR assignment and secondary structure analysis of the monomeric Pseudomonas biofilm-forming functional amyloid accessory protein FapA.

FapA is an accessory protein within the biofilm forming functional bacterial amyloid related fap-operon in Pseudomonas, and maybe a chaperone for FapC controlling its fibrillization. To allow further structural analysis, here we present a complete sequential assignment of 1Hamide, 13Cα, 13Cβ, and 15N NMR resonances for the functional form of the monomeric soluble FapA protein, comprising amino acids between 29 and 152. From these observed chemical shifts, the secondary structure propensities (SSPs) were determined. FapA predominantly adopts a random coil conformation, however, we also identified small propensities for α-helical and β-strand conformations. Notably, these observed SSPs are smaller compared to the ones we recently observed for the monomeric soluble FapC protein. These NMR results provide valuable insights into the activity of FapA in functional amyloid formation and regulation, that will also aid developing strategies targeting amyloid formation within biofilms and addressing chronic infections.

PHRONESIS: A One-Shot Approach for Sequential Assignment of Protein Resonances by Ultrafast MAS Solid-State NMR Spectroscopy.

Solid‐state NMR (ssNMR) spectroscopy has emerged as the method of choice to analyze the structural dynamics of fibrillar, membrane‐bound, and crystalline proteins that are recalcitrant to other structural techniques. Recently, 1H detection under fast magic angle spinning and multiple acquisition ssNMR techniques have propelled the structural analysis of complex biomacromolecules. However, data acquisition and resonance‐specific assignments remain a bottleneck for this technique. Here, we present a comprehensive multi‐acquisition experiment (PHRONESIS) that simultaneously generates up to ten 3D 1H‐detected ssNMR spectra. PHRONESIS utilizes broadband transfer and selective pulses to drive multiple independent polarization pathways. High selectivity excitation and de‐excitation of specific resonances were achieved by high‐fidelity selective pulses that were designed using a combination of an evolutionary algorithm and artificial intelligence. We demonstrated the power of this approach with microcrystalline U‐13C,15N GB1 protein, reaching 100 % of the resonance assignments using one data set of ten 3D experiments. The strategy outlined in this work opens up new avenues for implementing novel 1H‐detected multi‐acquisition ssNMR experiments to speed up and expand the application to larger biomolecular systems.

Conformational characterization of duplex DNA with solution-state NMR spectroscopy

DNA is the hereditary material for living organisms. Understanding the structural features of DNA is essential to comprehend various biological processes. This review outlines the application of solution-state NMR methods to study the structure and dynamics of duplex DNA. A brief overview of the context of NMR in the earlier days of nucleic acids research is discussed. Sample preparation methods and pertinent NMR measurables are introduced, followed by sequential resonance assignment protocol of DNA duplexes. A summary of results obtained from the application of NMR methodology to compare, contrast, and validate initial crystal crystallographic studies of DNA structure, especially the Drew-Dickerson dodecamer and A-tract sequences, is presented. This is followed by a survey of structural insights obtained in studies involving sequence-specific characteristics and mispairs. Results from relaxations experiments that reveal base pairs opening dynamics and short lived transient states of DNA is summarized. Overall, this review aims to introduce the atomistic characterization of DNA using NMR spectroscopy to a beginner level researcher.

Elucidation of a nutlin-derivative—HDM2 complex structure at the interaction site by NMR molecular replacement: A straightforward derivation

Protein—ligand complex structures are key in structure-based drug discovery, but their derivation largely relies on X-ray crystallography. While NMR is able to provide atomic resolution complex structures, traditional NMR structure calculation methods are too slow for drug discovery timelines. We recently developed the NMR molecular replacement (NMR2) method that substantially reduces the time needed to derive protein—ligand complex structures, mainly by bypassing the laborious protein sequential resonance assignment step. Here we show how we applied NMR2 to derive the structure of the protein HDM2 in complex with the small molecule caylin-1, an analog of nutlin, based on the HDM2—nutlin complex structure that was already derived by NMR2. This study illustrates how sparse information from a previous NMR2 structure elucidation can be employed to efficiently determine further protein-analog complex structures. We think NMR2 has the potential to become a major tool in structure-based drug discovery, especially when X-ray crystallography is difficult to implement.

NMR resonance assignments of human Atg3 in aqueous solution and bicelles.

Human Atg3 (hAtg3) is an E2-like enzyme that catalyzes the conjugation of LC3 family proteins to phosphatidylethanolamine (PE) lipids in the autophagosomal membrane during autophagy. The reaction product, LC3-PE, acts as a marker for autophagic cargo and is required for the effective construction of functional autophagosomes. However, the structural and molecular basis of this conjugation reaction remains elusive, at least in part, because of the absence of lipid bilayers in structural studies conducted to date. Here, we report a sequential resonance assignment for an hAtg3 construct both in aqueous solution and in bicelles. hAtg3 has 314 residues, and our construct lacks the unstructured region from residues 90 to 190. Our results demonstrate a structural rearrangement of hAtg3 N-terminus when it interacts with membranes.

What Your Crystal Structure Will Not Tell You about Enzyme Function.

ConspectusEnzyme function requires that enzyme structures be dynamic. Substrate binding, product release, and transition state stabilization typically involve different enzyme conformers. Furthermore, in multistep enzyme-catalyzed reactions, more than one enzyme conformation may be important for stabilizing different transition states. While X-ray crystallography provides the most detailed structural information of any current methodology, X-ray crystal structures of enzymes capture only those conformations that fit into the crystal lattice, which may or may not be relevant to function. Solution nuclear magnetic resonance (NMR) methods can provide an alternative approach to characterizing enzymes under nonperturbing and controllable conditions, allowing one to identify and localize dynamic processes that are important to function. However, many enzymes are too large for standard approaches to making sequential resonance assignments, a critical first step in analyzing and interpreting the wealth of information inherent in NMR spectra.This Account describes our long-standing NMR-based research into structural and dynamic aspects of function in the cytochrome P450 monooxygenase superfamily. These heme-containing enzymes typically catalyze the oxidation of unactivated C–H and C=C bonds in a multitude of substrates, often with complete regio- and stereospecificity. Over 600 000 genes in GenBank have been assigned to P450s, yet all known P450 structures exhibit a highly conserved and unique fold. This combination of functional and structural conservation with a vast substrate clientele, each substrate having multiple possible sites for oxidation, makes the P450s a unique target for understanding the role of enzyme structure and dynamics in determining a particular substrate–product combination. P450s are large by solution NMR standards, requiring us to develop specialized approaches for making sequential resonance assignments and interpreting the spectral changes that occur as a function of changing conditions (e.g., oxidation and spin state changes, ligand, substrate or effector binding). Solution conformations are characterized by the fitting of residual dipolar couplings (RDCs) measured for sequence-specifically assigned amide N–H correlations to alignment tensors optimized in the course of restrained molecular dynamics (MD) simulations. The conformational ensembles obtained by such RDC-restrained simulations, which we call “soft annealing”, are then tested by site-directed mutation and spectroscopic and activity assays for relevance. These efforts have gained us insights into cryptic conformational changes associated with substrate and redox partner binding that were not suspected from crystal structures, but were shown by subsequent work to be relevant to function. Furthermore, it appears that many of these changes can be generalized to P450s besides those that we have characterized, providing guidance for enzyme engineering efforts. While past research was primarily directed at the more tractable prokaryotic P450s, our current efforts are aimed at medically relevant human enzymes, including CYP17A1, CYP2D6, and CYP3A4.

Sequential backbone resonance assignment of AT-rich interaction domain of human BAF200.

BAF200 is a subunit of PBAF chromatin remodeling complex that contains an N-terminal AT-rich interaction domain (ARID). ARID domain in general has been shown to bind to the AT-rich DNA sequences. The human BAF200 ARID (~ 110 residues) has the potential to bind the DNA sequences with high affinity, however, the structure and the exact contribution of hBAF200 ARID in PBAF functions as well its DNA binding specificities have not been established. In this study, we have expressed and purified the hBAF200 ARID for NMR studies. We report the complete backbone 1H, 13C, and 15N chemical shift assignment and secondary structure of hBAF200 ARID domain.

Automated assignment of NMR spectra of macroscopically oriented proteins using simulated annealing

An automated technique for the sequential assignment of NMR backbone resonances of oriented protein samples has been developed and tested based on 15N-15N homonuclear exchange and spin-exchanged separated local-field spectra. By treating the experimental spectral intensity as a pseudopotential, the Monte-Carlo Simulated Annealing algorithm has been employed to seek lowest-energy assignment solutions over a large sampling space where direct enumeration would be unfeasible. The determined sequential assignments have been scored based on the positions of the crosspeaks resulting from the possible orders for the main peaks. This approach is versatile in terms of the parameters that can be specified to achieve the best-fit result. At a minimum the algorithm requires a continuous segment of the main-peak chemical shifts obtained from a uniformly labeled sample and a spin-exchanged experimental spectrum represented as a 2D matrix array. With selective labeling experiments, groups of chemical shifts corresponding to specific locations in the protein backbone can be fixed, thereby decreasing the sampling space. The output from the program consists of a list of top-score main peak assignments, which can be subjected to further scoring criteria until a consensus solution is found. The algorithm has first been tested on a synthetic spectrum with randomly generated chemical shifts and dipolar couplings for the main peaks. The original assignments have been successfully recovered for as many as 100 main peaks when residue-type information was used even in the presence of substantial spectral peak overlap. The algorithm was then applied to assigning two sets of experimental spectra to recover and confirm the previously established assignments in an automated fashion. For the 20-residue transmembrane domain of Pf1 coat protein reconstituted in magnetically aligned bicelles, the original assignment by Park et al. (2010) was recovered by the automated algorithm with additional input from 5 selectively labeled amino acid spectra. The second case considered was the 46 residue Pf1 bacteriophage from Thiriot et al. (2005) and Knox et al. (2010), of which 38 residues were fit. Automated fitting resulted in several possible assignments but not exactly the original assignment. By using a post-fitting filtering procedure based on the number of missed cross peaks and Pf1 helical structure, a consensus spectroscopic assignment is proposed covering 84% of the original assignment. While the automated assignment works best in spectra with well-resolved crosspeaks, it also tolerates substantial spectral crowding to yield reasonable assignments in the cases where ambiguity and degeneracy of possible assignment solutions are inevitable.

Structural Characterization of Fibrils from Recombinant Human Islet Amyloid Polypeptide by Solid-State NMR: The Central FGAILS Segment Is Part of the β-Sheet Core.

Amyloid deposits formed from islet amyloid polypeptide (IAPP) are a hallmark of type 2 diabetes mellitus and are known to be cytotoxic to pancreatic β-cells. The molecular structure of the fibrillar form of IAPP is subject of intense research, and to date, different models exist. We present results of solid-state NMR experiments on fibrils of recombinantly expressed and uniformly 13C, 15N-labeled human IAPP in the non-amidated, free acid form. Complete sequential resonance assignments and resulting constraints on secondary structure are shown. A single set of chemical shifts is found for most residues, which is indicative of a high degree of homogeneity. The core region comprises three to four β-sheets. We find that the central 23-FGAILS-28 segment, which is of critical importance for amyloid formation, is part of the core region and forms a β-strand in our sample preparation. The eight N-terminal amino acid residues of IAPP, forming a ring-like structure due to a disulfide bridge between residues C2 and C7, appear to be well defined but with an increased degree of flexibility. This study supports the elucidation of the structural basis of IAPP amyloid formation and highlights the extent of amyloid fibril polymorphism.

Solid-state NMR sequential assignment of an Amyloid-β(1-42) fibril polymorph.

The formation of fibrils of the amyloid-β (Aβ) peptide is considered to be a key event in the pathology of Alzheimer's disease (AD). The determination of a high-resolution structure of these fibrils is relevant for the understanding of the molecular basis of AD. In this work, we present the sequential resonance assignment of one of the polymorphs of Aβ(1-42) fibrils. We show that most of the protein is rigid, while a stretch of 4 residues (11-14) is not visible by solid-state NMR spectroscopy due to dynamics.

3次元NMR測定による変性蛋白質の信号連鎖帰属において問題となる化学シフト縮重を解く方法

3次元NMR測定による変性蛋白質の信号連鎖帰属において問題となる化学シフト縮重を解く方法

Simultaneous cross polarization to 13C and 15N with 1H detection at 60 kHz MAS solid-state NMR

We describe high resolution MAS solid-state NMR experiments that utilize 1H detection with 60kHz magic angle spinning; simultaneous cross-polarization from 1H to 15N and 13C nuclei; bidirectional cross-polarization between 13C and 15N nuclei; detection of both amide nitrogen and aliphatic carbon 1H; and measurement of both 13C and 15N chemical shifts through multi-dimensional correlation experiments. Three-dimensional experiments correlate amide 1H and alpha 1H selectively with 13C or 15N nuclei in a polypeptide chain. Two separate three-dimensional spectra correlating 1Hα/13Cα/1HN and 1HN/15N/1Hα are recorded simultaneously in a single experiment, demonstrating that a twofold savings in experimental time is potentially achievable. Spectral editing using bidirectional coherence transfer pathways enables simultaneous magnetization transfers between 15N, 13Cα(i) and 13C′(i−1), facilitating intra- and inter-residue correlations for sequential resonance assignment. Non-uniform sampling is integrated into the experiments, further reducing the length of experimental time.

Sequential backbone resonance assignments of the E. coli dihydrofolate reductase Gly67Val mutant: folate complex.

Occasionally, a mutation in an exposed loop region causes a significant change in protein function and/or stability. A single mutation Gly67Val of E. coli dihydrofolate reductase (DHFR) in the exposed CD loop is such an example. We have carried out the chemical shift assignments for H(N), N(H), C(α) and C(β) atoms of the Gly67Val mutant of E. coli DHFR complexed with folate at pH 7.0, 35°C, and then evaluated the H(N), N(H), C(α) and C(β) chemical shift changes caused by the mutation. The result indicates that, while the overall secondary structure remains the same, the single mutation Gly67Val causes site-specific conformational changes of the polypeptide backbone restricted around the adenosine-binding subdomain (residues 38-88) and not in the distant catalytic domain.

High resolution 4D HPCH experiment for sequential assignment of (13)C-labeled RNAs via phosphodiester backbone.

The three-dimensional structure determination of RNAs by NMR spectroscopy requires sequential resonance assignment, often hampered by assignment ambiguities and limited dispersion of 1H and 13C chemical shifts, especially of C4′/H4′. Here we present a novel through-bond 4D HPCH NMR experiment involving phosphate backbone where C4′–H4′ correlations are resolved along the 1H3′–31P spectral planes. The experiment provides high peak resolution and effectively removes ambiguities encountered during assignments. Enhanced peak dispersion is provided by the inclusion of additional 31P and 1H3′ dimensions and constant-time evolution of chemical shifts. High spectral resolution is obtained by using non-uniform sampling in three indirect dimensions. The experiment fully utilizes the isotopic 13C-labeling with evolution of C4′ carbons. Band selective 13C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the C4′–C3′ and C4′–C5′ homonuclear couplings. Multiple quantum line narrowing is employed to minimize sensitivity loses. The 4D HPCH experiment is verified and successfully applied to a non-coding 34-nt RNA consisting typical structure elements and a 14-nt RNA hairpin capped by cUUCGg tetraloop.

Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase.

We present solid-state NMR assignments of the N-terminal domain of the DnaB helicase from Helicobacter pylori (153 residues) in its microcrystalline form. We use a sequential resonance assignment strategy based on three-dimensional NMR experiments. The resonance assignments obtained are compared with automated resonance assignments computed with the ssFLYA algorithm. An analysis of the (13)C secondary chemical shifts determines the position of the secondary structure elements in this α-helical protein.

Protein resonance assignment at MAS frequencies approaching 100kHz: a quantitative comparison of J-coupling and dipolar-coupling-based transfer methods.

We discuss the optimum experimental conditions to obtain assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100 kHz. We present a systematic examination of the MAS dependence of the amide proton T 2' times and a site-specific comparison of T 2' at 93 kHz versus 60 kHz MAS frequency. A quantitative analysis of transfer efficiencies of building blocks, as they are used for typical 3D experiments, was performed. To do this, we compared dipolar-coupling and J-coupling based transfer steps. The building blocks were then combined into 3D experiments for sequential resonance assignment, where we evaluated signal-to-noise ratio and information content of the different 3D spectra in order to identify the best assignment strategy. Based on this comparison, six experiments were selected to optimally assign the model protein ubiquitin, solely using spectra acquired at 93 kHz MAS. Within 3 days of instrument time, the required spectra were recorded from which the backbone resonances have been assigned to over 96%.

Multiple acquisition of magic angle spinning solid-state NMR experiments using one receiver: Application to microcrystalline and membrane protein preparations

Solid-state NMR spectroscopy of proteins is a notoriously low-throughput technique. Relatively low-sensitivity and poor resolution of protein samples require long acquisition times for multidimensional NMR experiments. To speed up data acquisition, we developed a family of experiments called Polarization Optimized Experiments (POE), in which we utilized the orphan spin operators that are discarded in classical multidimensional NMR experiments, recovering them to allow simultaneous acquisition of multiple 2D and 3D experiments, all while using conventional probes with spectrometers equipped with one receiver. POE allow the concatenation of multiple 2D or 3D pulse sequences into a single experiment, thus potentially combining all of the aforementioned advances, boosting the capability of ssNMR spectrometers at least two-fold without the addition of any hardware. In this perspective, we describe the first generation of POE, such as dual acquisition MAS (or DUMAS) methods, and then illustrate the evolution of these experiments into MEIOSIS, a method that enables the simultaneous acquisition of multiple 2D and 3D spectra. Using these new pulse schemes for the solid-state NMR investigation of biopolymers makes it possible to obtain sequential resonance assignments, as well as distance restraints, in about half the experimental time. While designed for acquisition of heteronuclei, these new experiments can be easily implemented for proton detection and coupled with other recent advancements, such as dynamic nuclear polarization (DNP), to improve signal to noise. Finally, we illustrate the application of these methods to microcrystalline protein preparations as well as single and multi-span membrane proteins reconstituted in lipid membranes.

Easy and unambiguous sequential assignments of intrinsically disordered proteins by correlating the backbone 15N or 13C' chemical shifts of multiple contiguous residues in highly resolved 3D spectra.

Sequential resonance assignment strategies are typically based on matching one or two chemical shifts of adjacent residues. However, resonance overlap often leads to ambiguity in resonance assignments in particular for intrinsically disordered proteins. We investigated the potential of establishing connectivity through the three-bond couplings between sequentially adjoining backbone carbonyl carbon nuclei, combined with semi-constant time chemical shift evolution, for resonance assignments of small folded and larger unfolded proteins. Extended sequential connectivity strongly lifts chemical shift degeneracy of the backbone nuclei in disordered proteins. We show here that 3D (H)N(COCO)NH and (HN)CO(CO)NH experiments with relaxation-optimized multiple pulse mixing correlate up to seven adjacent backbone amide nitrogen or carbonyl carbon nuclei, respectively, and connections across proline residues are also obtained straightforwardly. Multiple, recurrent long-range correlations with ultra-high resolution allow backbone (1)H(N), (15)N(H), and (13)C' resonance assignments to be completed from a single pair of 3D experiments.