Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR.

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution.

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

TensorView for MATLAB: Visualizing tensors with Euler angle decoding.

TensorView for MATLAB is a GUI-based visualization tool for depicting second-rank Cartesian tensors as surfaces on three-dimensional molecular models. Both ellipsoid and ovaloid tensor display formats are supported, and the software allows for easy conversion of Euler angles from common rotation schemes (active, passive, ZXZ, and ZYZ conventions) with visual feedback. In addition, the software displays all four orientation-equivalent Euler angle solutions for the placement of a single tensor in the molecular frame and can report relative orientations of two tensors with all 16 orientation-equivalent Euler angle sets that relate them. The salient relations are derived and illustrated through several examples. TensorView for MATLAB expands and complements the earlier implementation of TensorView within the Mathematica programming environment and can be run without a MATLAB license. TensorView for MATLAB is available through github at https://github.com/LeoSvenningsson/TensorViewforMatlab, and can also be accessed directly via the NMRbox resource.

Ring Contraction of a Tungstacyclopentane Supported on Silica: Direct Conversion of Ethylene to Propylene.

The reaction of W(NAr)(13C4H8)(OSiPh3)2 (1) (NAr = 2,6-diisopropylphenylimido) with silica partially dehydroxylated at 700 °C (SiO2-700) is highly dependent on the reaction conditions. The primary product of this reaction is W(NAr)(13C4H8)(OSiPh3)(OSi(O-)3) (2) when the reaction is carried out in the dark. Grafting 1 onto SiO2-700 in ambient lab light results in the formation of 2, W(NAr)(13CH213CH2)(OSiPh3)(OSi(O-)3) (4), and one isomer of square-pyramidal W(NAr)(13CH213CH(13Me)13CH2)(OSiPh3)(OSi(O-)3) (3). Heating 2 to 85 °C for 6 h results in the formation of 3, 4, W(NAr)(13CH(13Me)13CH213CH2)(OSiPh3)(OSi(O-)3) (5), and W(NAr)((13CH2)213CH(13Me)(13CH2)2)(OSiPh3)(OSi(O-)3) (6). Photolysis of 2 with blue LEDs (λmax = 450 nm) produces 4, both isomers of 3, 5, and free ethylene. In the presence of excess ethylene and blue LED irradiation at 85 °C, 1/SiO2-700 catalyzes the direct conversion of ethylene to propylene.

Correlating Reaction Dynamics and Size Change during the Photomechanical Transformation of 9-Methylanthracene Single Crystals.

Photomechanical molecular crystals that expand under illumination could potentially be used as photon-powered actuators. In this study, we find that the use of high-quality single crystals of 9-methylanthracene (9MA) leads to more homogeneous reaction kinetics than that previously seen for polycrystalline samples, presumably due to a lower concentration of defects. Furthermore, simultaneous observation of absorbance and shape changes in single crystals revealed that the dimensional change mirrors the reaction progress, resulting in a smooth expansion of 7 % along the c-axis that is linearly correlated with reaction progress. The same expansion dynamics are highly reproducible across different single crystal samples. Organic single crystals exhibit well-defined linear expansions during 100 % photoconversion, suggesting that this class of solid-state phase change material could be used for actuation.

Moderated Basicity of Endohedral Amine Groups in an Octa-Cationic Self-Assembled Cage.

A self-assembled FeII4 L6 cage was synthesized with 12 internal amines in the cavity. The cage forms as the dodeca-ammonium salt, despite the cage carrying an overall 8+ charge at the metal centers, extracting protons from displaced water in the reaction. Despite this, the basicity of the internal amines is lower than their counterparts in free solution. The 12 amines have a sliding scale of basicity, with a ≈6 pKa unit difference between the first and last protons to be removed. This moderation of side-chain basicity in an active site is a hallmark of enzymatic catalysis.

Mutation of ßGln114 to Ala Alters the Stabilities of Allosteric States in Tryptophan Synthase Catalysis.

The tryptophan synthase (TS) bienzyme complexes found in bacteria, yeasts, and molds are pyridoxal 5'-phosphate (PLP)-requiring enzymes that synthesize l-Trp. In the TS catalytic cycle, switching between the open and closed states of the α- and ß-subunits via allosteric interactions is key to the efficient conversion of 3-indole-d-glycerol-3'-phosphate and l-Ser to l-Trp. In this process, the roles played by ß-site residues proximal to the PLP cofactor have not yet been fully established. ßGln114 is one such residue. To explore the roles played by ßQ114, we conducted a detailed investigation of the ßQ114A mutation on the structure and function of tryptophan synthase. Initial steady-state kinetic and static ultraviolet-visible spectroscopic analyses showed the Q to A mutation impairs catalytic activity and alters the stabilities of intermediates in the ß-reaction. Therefore, we conducted X-ray structural and solid-state nuclear magnetic resonance spectroscopic studies to compare the wild-type and ßQ114A mutant enzymes. These comparisons establish that the protein structural changes are limited to the Gln to Ala replacement, the loss of hydrogen bonds among the side chains of ßGln114, ßAsn145, and ßArg148, and the inclusion of waters in the cavity created by substitution of the smaller Ala side chain. Because the conformations of the open and closed allosteric states are not changed by the mutation, we hypothesize that the altered properties arise from the lost hydrogen bonds that alter the relative stabilities of the open (ßT state) and closed (ßR state) conformations of the ß-subunit and consequently alter the distribution of intermediates along the ß-subunit catalytic path.

Toho-1 ß-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam.

Backbone chemical shift assignments for the Toho-1 ß-lactamase (263 amino acids, 28.9 kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly 2H,13C,15N-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels. At the chemical level, titration with the non-ß-lactam ß-lactamase inhibitor avibactam is found to give chemical shift perturbations indicative of tight covalent binding that allow for mapping of the inhibitor binding site. At the structural level, protein secondary structure is predicted based on the backbone chemical shifts and protein residue sequence using TALOS-N and found to agree well with structural characterization from X-ray crystallography. At the dynamical level, model-free analysis of 15N relaxation data at a single field of 16.4 T reveals well-ordered structures for the ligand-free and avibactam-bound enzymes with generalized order parameters of ~ 0.85. Complementary relaxation dispersion experiments indicate that there is an escalation in motions on the millisecond timescale in the vicinity of the active site upon substrate binding. The combination of high rigidity on short timescales and active site flexibility on longer timescales is consistent with hypotheses for achieving both high catalytic efficiency and broad substrate specificity: the induced active site dynamics allows variously sized substrates to be accommodated and increases the probability that the optimal conformation for catalysis will be sampled.

Backbone assignments and conformational dynamics in the S. typhimurium tryptophan synthase α-subunit from solution-state NMR.

Backbone assignments for the isolated α-subunit of Salmonella typhimurium tryptophan synthase (TS) are reported based on triple resonance solution-state NMR experiments on a uniformly 2H,13C,15N-labeled sample. From the backbone chemical shifts, secondary structure and random coil index order parameters (RCI-S2) are predicted. Titration with the 3-indole-D-glycerol 3'-phosphate analog, N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), leads to chemical shift perturbations indicative of conformational changes from which an estimate of the dissociation constant is obtained. Comparisons of the backbone chemical-shifts, RCI-S2 values, and site-specific relaxation times with and without F9 reveal allosteric changes including modulation in secondary structures and loop rigidity induced upon ligand binding. A comparison is made to the X-ray crystal structure of the α-subunit in the full TS αßßα bi-enzyme complex and to two new X-ray crystal structures of the isolated TS α-subunit reported in this work.

Non-Uniform Sampling in NMR Spectroscopy and the Preservation of Spectral Knowledge in the Time and Frequency Domains.

The increased sensitivity under weighted non-uniform sampling (NUS) is demonstrated and quantified using Monte Carlo simulations of nuclear magnetic resonance (NMR) time- and frequency-domain signals. The concept of spectral knowledge is introduced and shown to be superior to the frequency-domain signal-to-noise ratio for assessing the quality of NMR data. Two methods for rigorously preserving spectral knowledge and the time-domain NUS knowledge enhancement upon transformation to the frequency domain are demonstrated, both theoretically and numerically. The first, non-uniform weighted sampling using consistent root-mean-square noise, is applicable to data sampled on the Nyquist grid, whereas the second, the block Fourier transform using consistent root-mean-square noise, can be used to transform time-domain data acquired with arbitrary, off-grid NUS.

Cofactor-Mediated Nucleophilic Substitution Catalyzed by a Self-Assembled Holoenzyme Mimic.

A self-assembled Fe4L6 cage is capable of co-encapsulating multiple carboxylic acid containing guests in its cavity, and these acids can act as cofactors for cage-catalyzed nucleophilic substitutions. The kinetics of the substitution reaction depend on the size, shape, and binding affinity of each of the components, and small structural changes in guest size can have large effects on the reaction. The host is quite promiscuous and is capable of binding multiple guests with micromolar binding affinities while retaining the ability to effect turnover and catalysis. Substrate binding modes vary widely, from simple 1:1 complexes to 1:2 complexes that can show either negative or positive cooperativity, depending on the guest. The molecularity of the dissociative substitution reaction varies, depending on the electrophile leaving group, acid cofactor, and nucleophile size: small changes in the nature of substrate can have large effects on reaction kinetics, all controlled by selective molecular recognition in the cage interior.

Direct dynamic nuclear polarization of 15N and 13C spins at 14.1 T using a trityl radical and magic angle spinning.

We investigate solid-state dynamic nuclear polarization of 13C and 15N nuclei using monoradical trityl OX063 as a polarizing agent in a magnetic field of 14.1 T with magic angle spinning at ∼100 K. We monitored the field dependence of direct 13C and 15N polarization for frozen [13C, 15N] urea and achieved maximum absolute enhancement factors of 240 and 470, respectively. The field profiles are consistent with polarization of 15N spins via either the solid effect or the cross effect, and polarization of 13C spins via a combination of cross effect and solid effect. For microcrystalline, 15N-enriched tryptophan synthase sample containing trityl radical, a 1500-fold increase in 15N signal was observed under microwave irradiation. These results show the promise of trityl radicals and their derivatives for direct polarization of low gamma, spin-½ nuclei at high magnetic fields and suggest a novel approach for selectively polarizing specific moieties or for polarizing systems which have low levels of protonation.

TensorView: A software tool for displaying NMR tensors.

The representation of nuclear magnetic resonance (NMR) tensors as surfaces on three-dimensional molecular models is an information-rich presentation that highlights the geometric relationship between tensor principal components and the underlying molecular and electronic structure. Here, we describe a new computational tool, TensorView, for depicting NMR tensors on the molecular framework. This package makes use of the graphical interface and built-in molecular display functionality present within the Mathematica programming environment and is robust for displaying tensor properties from a broad range of commercial and user-specific computational chemistry packages. Two mathematical forms for representing tensor interaction surfaces are presented, the popular ellipsoidal construct and the more technically correct "ovaloid" form. Examples are provided for chemical shielding and shift tensors, dipole-dipole and quadrupolar couplings, and atomic anisotropic displacement parameters (thermal ellipsoids) derived from NMR crystallography.

Predicting anisotropic thermal displacements for hydrogens from solid-state NMR: a study on hydrogen bonding in polymorphs of palmitic acid.

The hydrogen-bonding environments at the COOH moiety in eight polycrystalline polymorphs of palmitic acid are explored using solid-state NMR. Although most phases have no previously reported crystal structure, measured 13C chemical shift tensors for COOH moieties, combined with DFT modeling establish that all phases crystallize with a cyclic dimer (R22(8)) hydrogen bonding arrangement. Phases A2, Bm and Em have localized OH hydrogens while phase C has a dynamically disordered OH hydrogen. The phase designated As is a mix of five forms, including 27.4% of Bm and four novel phases not fully characterized here due to insufficient sample mass. For phases A2, Bm, Em, and C the anisotropic uncertainties in the COOH hydrogen atom positions are established using a Monte Carlo sampling scheme. Sampled points are retained or rejected at the ±1σ level based upon agreement of DFT computed 13COOH tensors with experimental values. The collection of retained hydrogen positions bear a remarkable resemblance to the anisotropic displacement parameters (i.e. thermal ellipsoids) from diffraction studies. We posit that this similarity is no mere coincidence and that the two are fundamentally related. The volumes of NMR-derived anisotropic displacement ellipsoids for phases with localized OH hydrogens are 4.1 times smaller than those derived from single crystal X-ray diffraction and 1.8 times smaller than the volume of benchmark single crystal neutron diffraction values.

Visualizing the tunnel in tryptophan synthase with crystallography: Insights into a selective filter for accommodating indole and rejecting water.

Four new X-ray structures of tryptophan synthase (TS) crystallized with varying numbers of the amphipathic N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) molecule are presented. These structures show one of the F6 ligands threaded into the tunnel from the ß-site and reveal a distinct hydrophobic region. Over this expanse, the interactions between F6 and the tunnel are primarily nonpolar, while the F6 phosphoryl group fits into a polar pocket of the ß-subunit active site. Further examination of TS structures reveals that one portion of the tunnel (T1) binds clusters of water molecules, whereas waters are not observed in the nonpolar F6 binding region of the tunnel (T2). MD simulation of another TS structure with an unobstructed tunnel also indicates the T2 region of the tunnel excludes water, consistent with a dewetted state that presents a significant barrier to the transfer of water into the closed ß-site. We conclude that hydrophobic molecules can freely diffuse between the α- and ß-sites via the tunnel, while water does not. We propose that exclusion of water serves to inhibit reaction of water with the α-aminoacrylate intermediate to form ammonium ion and pyruvate, a deleterious side reaction in the αß-catalytic cycle. Finally, while most TS structures show ßPhe280 partially blocking the tunnel between the α- and ß-sites, new structures show an open tunnel, suggesting the flexibility of the ßPhe280 side chain. Flexible docking studies and MD simulations confirm that the dynamic behavior of ßPhe280 allows unhindered transfer of indole through the tunnel, therefore excluding a gating role for this residue.

Measuring and Modeling Highly Accurate 15 N Chemical Shift Tensors in a Peptide.

NMR studies measuring chemical shift tensors are increasingly being employed to assign structure in difficult-to-crystallize solids. For small organic molecules, such studies usually focus on 13 C sites, but proteins and peptides are more commonly described using 15 N amide sites. An important and often neglected consideration when measuring shift tensors is the evaluation of their accuracy against benchmark standards, where available. Here we measure 15 N tensors in the dipeptide glycylglycine at natural abundance using the slow-spinning FIREMAT method with SPINAL-64 decoupling. The accuracy of these 15 N tensors is evaluated by comparing to benchmark single crystal NMR 15 N measurements and found to be statistically indistinguishable. These FIREMAT experimental results are further used to evaluate the accuracy of theoretical predictions of tensors from four different density functional theory (DFT) methods that include lattice effects. The best theoretical approach provides a root mean square (rms) difference of ±3.9 ppm and is obtained from a fragment-based method and the PBE0 density functional.

Catalytic roles of ßLys87 in tryptophan synthase: (15)N solid state NMR studies.

The proposed mechanism for tryptophan synthase shows ßLys87 playing multiple catalytic roles: it bonds to the PLP cofactor, activates C4' for nucleophilic attack via a protonated Schiff base nitrogen, and abstracts and returns protons to PLP-bound substrates (i.e. acid-base catalysis). ε-¹5N-lysine TS was prepared to access the protonation state of ßLys87 using ¹5N solid-state nuclear magnetic resonance (SSNMR) spectroscopy for three quasi-stable intermediates along the reaction pathway. These experiments establish that the protonation state of the ε-amino group switches between protonated and neutral states as the ß-site undergoes conversion from one intermediate to the next during catalysis, corresponding to mechanistic steps where this lysine residue has been anticipated to play alternating acid and base catalytic roles that help steer reaction specificity in tryptophan synthase catalysis. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. Guest Editors: Andrea Mozzarelli and Loredano Pollegioni.

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity.

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5'-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography-the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry-to interrogate a carbanionic/quinonoid intermediate analogue in the ß-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4' of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for ß-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.

Benchmark fragment-based (1)H, (13)C, (15)N and (17)O chemical shift predictions in molecular crystals.

The performance of fragment-based ab initio(1)H, (13)C, (15)N and (17)O chemical shift predictions is assessed against experimental NMR chemical shift data in four benchmark sets of molecular crystals. Employing a variety of commonly used density functionals (PBE0, B3LYP, TPSSh, OPBE, PBE, TPSS), we explore the relative performance of cluster, two-body fragment, and combined cluster/fragment models. The hybrid density functionals (PBE0, B3LYP and TPSSh) generally out-perform their generalized gradient approximation (GGA)-based counterparts. (1)H, (13)C, (15)N, and (17)O isotropic chemical shifts can be predicted with root-mean-square errors of 0.3, 1.5, 4.2, and 9.8 ppm, respectively, using a computationally inexpensive electrostatically embedded two-body PBE0 fragment model. Oxygen chemical shieldings prove particularly sensitive to local many-body effects, and using a combined cluster/fragment model instead of the simple two-body fragment model decreases the root-mean-square errors to 7.6 ppm. These fragment-based model errors compare favorably with GIPAW PBE ones of 0.4, 2.2, 5.4, and 7.2 ppm for the same (1)H, (13)C, (15)N, and (17)O test sets. Using these benchmark calculations, a set of recommended linear regression parameters for mapping between calculated chemical shieldings and observed chemical shifts are provided and their robustness assessed using statistical cross-validation. We demonstrate the utility of these approaches and the reported scaling parameters on applications to 9-tert-butyl anthracene, several histidine co-crystals, benzoic acid and the C-nitrosoarene SnCl2(CH3)2(NODMA)2.