Solution NMR Spectroscopy Research Articles

Bisisocyanide cyclometallated platinum(II) complexes: synthesis, structure, photophysical properties, and mechanochromic behavior

A series of cyclometallated platinum(II) complexes [Pt(ppy){CNAr}2]X with two isocyanide ligands (Hppy = 2-phenylpyridine, Ar = C6H2-2,4,6-Me3, C6H3-2-Cl-6-Me , C6H3-2,6-Cl2, C6H4-4-NMe2, C6H4-4-Me, C6H4 4-Cl, C6H4-4-Br, C6H4-4-I, C6H4-4-CF3, C6H4-3-CF3; X = BF4, OTf) was synthesized by the reaction of the [{Pt(ppy)Cl}2] dimer with isocyanides (yield 52-70%). The structure of the resulting complexes was determined using mass spectrometry, 1H, 13C{1H}, 195Pt{1H}, 1H-1H COSY, 1H-1H NOESY, 1H-13C HSQC, and 1H-13C HMBC NMR spectroscopy in solution and solid-state CP/MAS 13C and 195Pt NMR spectroscopy, IR spectroscopy and X-ray diffraction analysis in the solid phase. The photophysical properties of the obtained complexes in the solid phase and the mechanochromic luminescence behavior were studied. In the solid phase, all synthesized compounds phosphoresce in the green or orange range of visible light, while photoluminescence quantum yields reach 26%. Green phosphors exhibit a reversible mechanochromic luminescence change achieved by mechanical grinding (green to orange) and solvent adsorption (orange to green).

NMR Studies of Aromatic Ring Flips to Probe Conformational Fluctuations in Proteins.

Aromatic residues form a significant part of the protein core, where they make tight interactions with multiple surrounding side chains. Despite the dense packing of internal side chains, the aromatic rings of phenylalanine and tyrosine residues undergo 180° rotations, or flips, which are mediated by transient and large-scale "breathing" motions that generate sufficient void volume around the aromatic ring. Forty years after the seminal work by Wagner and Wüthrich, NMR studies of aromatic ring flips are now undergoing a renaissance as a powerful means of probing fundamental dynamic properties of proteins. Recent developments of improved NMR methods and isotope labeling schemes have enabled a number of advances in addressing the mechanisms and energetics of aromatic ring flips. The nature of the transition states associated with ring flips can be described by thermodynamic activation parameters, including the activation enthalpy, activation entropy, activation volume, and also the isothermal volume compressibility of activation. Consequently, it is of great interest to study how ring flip rate constants and activation parameters might vary with protein structure and external conditions like temperature and pressure. The field is beginning to gather such data for aromatic residues in a variety of environments, ranging from surface exposed to buried. In the future, the combination of solution and solid-state NMR spectroscopy together with molecular dynamics simulations and other computational approaches is likely to provide detailed information about the coupled dynamics of aromatic rings and neighboring residues. In this Perspective, we highlight recent developments and provide an outlook toward the future.

Mechanisms by which small molecules of diverse chemotypes arrest Sec14 lipid transfer activity

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood. Herein, we apply an integrative structural biology approach to investigate interactions of the yeast PITP Sec14 with small-molecule inhibitors (SMIs) of its phospholipid exchange cycle. Using a combination of X-ray crystallography, solution NMR spectroscopy, and atomistic MD simulations, we dissect how SMIs compete with native Sec14 phospholipid ligands and arrest phospholipid exchange. Moreover, as Sec14 PITPs represent new targets for the development of next-generation antifungal drugs, the structures of Sec14 bound to SMIs of diverse chemotypes reported in this study will provide critical information required for future structure-based design of next-generation lead compounds directed against Sec14 PITPs of virulent fungi.

N-Coordinated tellurenium(II) and telluronium(IV) cations: synthesis, structure and hydrolysis.

A set of N-coordinated tellurium(II) compounds containing either C,N-chelating ligands CNR (where CN = 2-(RNCH)C6H4, R = tBu or Dipp; Dipp = 2,6-iPr2C6H3) or N,C,N pincer ligands NCNR (where NCN = 2,6-(RNCH)2C6H4, R = tBu or Dipp) were synthesized. In the case of C,N-chelated compounds, the reaction of CNDippLi with Te(dtc)2 (where dtc = Et2NCS2) in a 1 : 1 molar ratio smoothly provided the carbamate CNDippTe(dtc) which upon treatment with 2 eq. of HCl provided the chloride CNDippTeCl. In contrast, the analogous conversion of NCNRLi with Te(dtc)2 surprisingly furnished ionic bromides [NCNRTe]Br as a result of the exchange of dtc by Br coming from nBuBr present in the reaction mixture. Furthermore, the reaction of CNDippTeCl or [NCNRTe]Br with silver salts AgX (X = OTf or SbF6) provided the expected tellurenium cations [CNDippTe]SbF6 and [NCNRTe]X. To further increase the Lewis acidity of the central atom, the oxidation of selected compounds with 1 eq. of SO2Cl2 was examined yielding stable compounds [CNtBuTeCl2]X and [NCNtBuTeCl2]X. The oxidation of the Dipp substituted compounds proved to be more challenging and an excess of SO2Cl2 was necessary to obtain the oxidized products [CNDippTeCl2]SbF6 and [NCNDippTeCl2]SbF6, which could solely be characterized in solution. Compounds [CNtBuTeCl2]OTf and [NCNtBuTeCl2]OTf were shown to undergo a controlled hydrolysis to the corresponding telluroxanes. All compounds were studied by multinuclear NMR spectroscopy in solution and for selected compounds solid state 125Te NMR spectroscopy and single-crystal X-ray diffraction analysis were performed. The Lewis acidity of the studied cations was examined by the Gutmann-Beckett method using Et3PO as the probing agent. The Te-N chalcogen bonding situation of selected compounds has also been examined computationally by a set of real-space bonding indicators.

Helix-forming aliphatic homo-δ-peptide foldamers based on the conformational restriction effects of cyclopropane.

Considerable effort has been directed toward developing artificial peptide-based foldamers. However, detailed structural analysis of δ-peptide foldamers consisting of only aliphatic δ-amino acids has not been reported. Herein, we rationally designed and stereoselectively synthesized aliphatic homo-δ-peptides forming a stable helical structure by using a chiral cyclopropane δ-amino acid as a monomer unit. Structural analysis of the homo-δ-peptides using circular dichroism, infrared, and NMR spectroscopy indicated that they form a stable 14-helical structure in solution. Furthermore, we successfully conducted X-ray crystallographic analysis of the homo-δ-peptides, demonstrating a right-handed 14-helical structure. This helical structure of the crystal was consistent with those predicted by theoretical calculations and those obtained based on NMR spectroscopy in solution. This stable helical structure is due to the effective restriction of the backbone conformation by the structural characteristics of cyclopropane. This work reports the first example of aliphatic homo-δ-peptide foldamers having a stable helical structure both in the solution and crystal states.

Analysis of coordinated NMR chemical shifts to map allosteric regulatory networks in proteins.

The exquisite sensitivity of the NMR chemical shift to local environment makes it an ideal probe to assess atomic level perturbations in proteins of all sizes and structural compositions. Recent advances in solution and solid-state NMR spectroscopy of biomolecules have leveraged the chemical shift to report on short- and long-range couplings between individual amino acids to establish "networks" of residues that form the basis of allosteric pathways that transmit chemical signals through the protein matrix to induce functional responses. The simple premise that thermodynamically and functionally coupled regions of a protein (i.e. active and allosteric sites) should be reciprocally sensitive to structural or dynamic perturbations has enabled NMR spectroscopy, the premier method for molecular resolution of protein structural fluctuations, to occupy a place at the forefront of investigations into protein allostery. Here, we detail several key methods of NMR chemical shift analysis to extract mechanistic information about long-range chemical signaling in a protein, focusing on practical methodological aspects and the circumstances under which a given approach would be relevant. We also detail some of the experimental considerations that should be made when applying these methods to specific protein systems.

Steady-State Free Precession sequences for high and low field NMR spectroscopy in solution: Challenges and opportunities

The receptivity of NMR spectroscopy is low when compared to other techniques. Historically, increasing the strength of the static magnetic field has been the major approach to increase NMR sensitivity. In recent years several polarization transfer protocols have been used to enhance the signal-to-noise ratio (SNR), although they require special accessories and/or sample preparation. In this paper, we consider both the challenges and opportunities of steady-state free precession (SSFP) pulse sequences as a simple and efficient alternative to enhance SNR, in standard high-resolution and benchtop low-resolution NMR spectrometers. The maximum gain in these sequences is obtained with the shortest time between the pulses (Tp). However, when Tp<T2, the SSFP signal contains FID and echo components which lead to phase, intensity, and truncation artifacts on spectra obtained by Fast Fourier transform (FT). Several phase alternation SSFP sequences were used to cancel the echo component and minimize these problems in the FT spectra. Krylov base diagonalization method (KBDM) was used to eliminate the phase and truncation problems in spectra acquired by SSFP pulse sequences and can be a viable alternative to FT. The experiments were performed in high and low resolution (bench top) NMR spectrometers and significant enhancements in SNR of low receptivity nuclei such as 13C and 15N could be achieved. The SSFP sequences were also shown to enhance SNR in nuclei with high receptivity such as 19F and 31P, in very dilute samples, as is common in environmental and biological samples.

Surface electrostatics dictate RNA-binding protein CAPRIN1 condensate concentration and hydrodynamic properties

Biomolecular condensates concentrate proteins, nucleic acids, and small molecules and play an essential role in many biological processes. Their formation is tuned by a balance between energetically favorable and unfavorable contacts, with charge-charge interactions playing a central role in some systems. The positively charged intrinsically disordered carboxy-terminal region of the RNA-binding protein CAPRIN1 is one such example, phase separating upon addition of negatively charged ATP or high concentrations of sodium chloride (NaCl). Using solution NMR spectroscopy, we measured residue-specific near-surface electrostatic potentials (ϕENS) of CAPRIN1 along its NaCl-induced phase separation trajectory to compare with those obtained using ATP. In both cases, electrostatic shielding decreases ϕENS values, yet surface potentials of CAPRIN1 in the two condensates can be different, depending on the amount of NaCl or ATP added. Our results establish that even small differences in ϕENS can significantly affect the level of protein enrichment and the mechanical properties of the condensed phase, leading, potentially, to the regulation of biological processes.

Mechanism of Alkyne Hydroarylation Catalyzed by (P,C)-Cyclometalated Au(III) Complexes.

Over the last 5-10 years, gold(III) catalysis has developed rapidly. It often shows complementary if not unique features compared to gold(I) catalysis. While recent work has enabled major synthetic progress in terms of scope and efficiency, very little is yet known about the mechanism of Au(III)-catalyzed transformations and the relevant key intermediates have rarely been authenticated. Here, we report a detailed experimental/computational mechanistic study of the recently reported intermolecular hydroarylation of alkynes catalyzed by (P,C)-cyclometalated Au(III) complexes. The cationic (P,C)Au(OAcF)+ complex (OAcF = OCOCF3) was authenticated by mass spectrometry (MS) in the gas phase and multi-nuclear NMR spectroscopy in solution at low temperatures. According to density functional theory (DFT) calculations, the OAcF moiety is κ2-coordinated to gold in the ground state, but the corresponding κ1-forms featuring a vacant coordination site sit only slightly higher in energy. Side-on coordination of the alkyne to Au(III) then promotes nucleophilic addition of the arene. The energy profiles for the reaction between trimethoxybenzene (TMB) and diphenylacetylene (DPA) were computed by DFT. The activation barrier is significantly lower for the outer-sphere pathway than for the alternative inner-sphere mechanism involving C-H activation of the arene followed by migratory insertion. The π-complex of DPA was characterized by MS. An unprecedented σ-arene Au(III) complex with TMB was also authenticated both in the gas phase and in solution. The cationic complexes [(P,C)Au(OAcF)]+ and [(P,C)Au(OAcF)(σ-TMB)]+ stand as active species and off-cycle resting state during catalysis, respectively. This study provides a rational basis for the further development of Au(III) catalysis based on π-activation.

Structural insights into acetylated histone ligand recognition by the BDP1 bromodomain of Plasmodium falciparum

Plasmodium falciparum requires a two-host system, moving between Anopheles mosquito and humans, to complete its life cycle. To overcome such dynamic growth conditions its histones undergo various post-translational modifications to regulate gene expression. The P. falciparum Bromodomain Protein 1 (PfBDP1) has been shown to interact with acetylated lysine modifications on histone H3 to regulate the expression of invasion-related genes. Here, we investigated the ability of the PfBDP1 bromodomain to interact with acetyllsyine modifications on additional core and variant histones. A crystal structure of the PfBDP1 bromodomain (PfBDP1-BRD) reveals it contains the conserved bromodomain fold, but our comparative analysis between the PfBDP1-BRD and human bromodomain families indicates it has a unique binding mechanism. Solution NMR spectroscopy and ITC binding assays carried out with acetylated histone ligands demonstrate that it preferentially recognizes tetra-acetylated histone H4, and we detected weaker interactions with multi-acetylated H2A.Z in addition to the previously reported interactions with acetylated histone H3. Our findings indicate PfBDP1 may play additional roles in the P. falciparum life cycle, and the distinctive features of its bromodomain binding pocket could be leveraged for the development of new therapeutic agents to help overcome the continuously evolving resistance of P. falciparum against currently available drugs.

1H, 13C, 15N backbone resonance assignment of apo and ADP-ribose bound forms of the macro domain of Hepatitis E virus through solution NMR spectroscopy

The genome of Hepatitis E virus (HEV) is 7.2 kilobases long and has three open reading frames. The largest one is ORF1, encoding a non-structural protein involved in the replication process, and whose processing is ill-defined. The ORF1 protein is a multi-modular protein which includes a macro domain (MD). MDs are evolutionarily conserved structures throughout all kingdoms of life. MDs participate in the recognition and removal of ADP-ribosylation, and specifically viral MDs have been identified as erasers of ADP-ribose moieties interpreting them as important players at escaping the early stages of host-immune response. A detailed structural analysis of the apo and bound to ADP-ribose state of the native HEV MD would provide the structural information to understand how HEV MD is implicated in virus-host interplay and how it interacts with its intracellular partner during viral replication. In the present study we present the high yield expression of the native macro domain of HEV and its analysis by solution NMR spectroscopy. The HEV MD is folded in solution and we present a nearly complete backbone and sidechains assignment for apo and bound states. In addition, a secondary structure prediction by TALOS + analysis was performed. The results indicated that HEV MD has a α/β/α topology very similar to that of most viral macro domains.

Stimuli-Induced Reversible Transformation between Decanuclear and Pentanuclear Gold(I) Sulfido Complexes.

Decanuclear and pentanuclear gold(I) sulfido complexes of phenanthrene- and dibenzothiophene-based diphosphine ligands were synthesized and characterized. Unprecedented stimuli-induced reversible transformation between decanuclear and pentanuclear gold(I) sulfido complexes was observed, which could be readily monitored by NMR and UV-vis absorption spectroscopy in solution. Remarkably, the decanuclear gold(I) sulfido complex (Au10-LPh) was found to show a highly reversible transformation process, which is stable for over 10 successive cycles in solution. The stimuli-induced reversible transformation behavior of the gold(I) sulfido complexes was found to depend on the P-P bite distance of the bidentate phosphine ligands.

Real-Time 1H and 31P NMR spectroscopy of the copolymerization of cyclic phosphoesters and trimethylene carbonate reveals transesterification from gradient to random copolymers

Copolymerization is the general approach to combine properties of different polymers in a single material. We prepared the first trimethylene carbonate (TMC) phosphoester (PE) copolymers by statistical copolymerization using either stannous octanoate or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as the respective catalyst. Variation of comonomer feed, reaction temperature and solvent, resulted in a library of copolymers with molar masses of up to 83,000 g/mol and moderate molar mass dispersities (<1.8). The monomer consumptions during the copolymerizations were followed by real-time 1H and 31P NMR spectroscopy in solution and in the bulk. As ring-opening copolymerization of cyclic esters often leads to gradient copolymers, also TMC and PE copolymerizations resulted in gradient copolymers, i.e. P(TMC-grad-PE)s, with different gradient strength depending on the copolymerization conditions. If a suitable transesterification catalyst like stannous octanoate was used, randomization of the gradient copolymer occurred. The 31P NMR shift of incorporated phosphoester units was used as a probe to monitor the randomization of the gradient copolymers in real-time by 31P NMR spectroscopy. The P-comonomer acts as an ideal NMR probe for in situ monitoring the changes of the copolymers’ microstructure during the transesterification by following the evolution of diads and triads. This paper focusses on the randomization procedure and gives detailed information about the copolymer structure and the kinetics of the copolymerization. The data give the possibility to understand randomization processes on a molecular scale and to fine-tune properties of polycarbonate-polyphosphoester copolymers in future applications.

Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2.

NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH–N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein.

Mapping the per-residue surface electrostatic potential of CAPRIN1 along its phase-separation trajectory

Electrostatic interactions and charge balance are important for the formation of biomolecular condensates involving proteins and nucleic acids. However, a detailed, atomistic picture of the charge distribution around proteins during the phase-separation process is lacking. Here, we use solution NMR spectroscopy to measure residue-specific near-surface electrostatic potentials (ϕENS) of the positively charged carboxyl-terminal intrinsically disordered 103 residues of CAPRIN1, an RNA-binding protein localized to membraneless organelles playing an important role in messenger RNA (mRNA) storage and translation. Measured ϕENS values have been mapped along the adenosine triphosphate (ATP)-induced phase-separation trajectory. In the absence of ATP, ϕENS values for the mixed state of CAPRIN1 are positive and large and progressively decrease as ATP is added. This is coupled to increasing interchain interactions, particularly between aromatic-rich and arginine-rich regions of the protein. Upon phase separation, CAPRIN1 molecules in the condensed phase are neutral (ϕENS [Formula: see text] 0 mV), with ∼five molecules of ATP associated with each CAPRIN1 chain. Increasing the ATP concentration further inverts the CAPRIN1 electrostatic potential, so that molecules become negatively charged, especially in aromatic-rich regions, leading to re-entrance into a mixed phase. Our results collectively show that a subtle balance between electrostatic repulsion and interchain attractive interactions regulates CAPRIN1 phase separation and provides insight into how nucleotides, such as ATP, can induce formation of and subsequently dissolve protein condensates.

Multilayer protein corona on gold nanorod surface: First evidence of soft corona protein-protein interactions using solution NMR spectroscopy

The nanoparticle surfaces interact readily with biomolecules, including proteins forming a corona. The protein layer that directly interacts with the nanoparticle surface is usually explored. Non-covalent interactions of proteins in the second layer have not been elucidated. We report for the first-time multilayer protein-protein interactions in ubiquitin corona on gold nanorod surface. The conformational dynamics of the protein, ubiquitin, on the surface of gold nanorods were probed at the molecular level using two-dimensional solution nuclear magnetic resonance spectroscopy. The interaction of Ubiquitin with gold nanorods was characterized by UV–visible spectroscopy, dynamic light scattering, and zeta-potential measurements. The residues of Ubiquitin that show significant perturbations fall into two categories: (a) the residues that directly interact with the surface of gold nanorods forming hard corona (primary interaction) and (b) the residues that are forming soft corona (secondary interaction). The formation of hard corona on the surface of the nanorod takes place even at a low concentration of the protein. The protein does not undergo any changes in its secondary or tertiary structure. As the protein concentration is increased, the hard corona, which is off mutually interconvertible multiple conformers, undergoes conformational reorientation to maximize its interaction with the nanorod surface. The study reveals electrostatically driven weak protein-nanorod interactions. Further, the study reveals two different processes, one involving the direct interaction with nanorods and the other involving protein corona formation in the protein-nanomaterial system. The study, for the first time, explores multilayer protein corona on a nanoparticle surface and provides new insights into the mechanisms involving protein aggregation in the presence of the nanoparticles.

New Phosphonite Ligands with High Steric Demand and Low Basicity: Synthesis, Structural Properties and Cyclometalated Complexes of Pt(II)

Two phosphonite ligands bearing the highly sterically demanding 2,6-bis (2,6-dimethylphenyl)phenyl group (ArXyl2), PArXyl2(OPhNO2)2 and PArXyl2(OPhNO2,Me)2, were prepared from the parent dihalophosphines PArXyl2X2 (X = Cl, Br) and the corresponding phenols, 4-nitrophenol and 4-nitro-2,6-dimethylphenol, respectively. DFT methods were used to examine their structural features and to determine three steric descriptors, namely the Tolman cone angle, the percentage of buried volume, and the percentage of the coordination sphere protected by the ligand. A comparison with the related terphenyl phosphines is also provided. Reactions of PArXyl2(OPhNO2)2 and PArXyl2(OPhNO2,Me)2 with several Pt(II) precursors were investigated, revealing a high tendency of both phosphonites to undergo C-H activation processes and generate five- or six-membered cyclometalated structures. The coordination chemistry of the new ligands was explored with isolation, among others, of three carbonyl complexes, 1-3∙CO, and the triphenylphosphine adduct 3∙PPh3. X-ray diffraction methods permitted the determination of the solid-state structures of the mononuclear methyl carbonyl complex 1∙CO, the dinuclear chloride-bridged complex 2 and the doubly cyclometalated complex 3∙SMe2, including the conformations adopted by the ligands upon coordination. All of the new compounds were characterized by multinuclear NMR spectroscopy in solution.

Proton TOCSY NMR relaxation rates quantitate protein side chain mobility in the Pin1 WW domain

Protein side chain dynamics play a vital role in many biological processes, but differentiating mobile from rigid side chains remains a technical challenge in structural biology. Solution NMR spectroscopy is ideally suited for this but suffers from limited signal-to-noise, signal overlap, and a need for fractional 13C or 2H labeling. Here we introduce a simple strategy measuring initial 1H relaxation rates during a 1H TOCSY sequence like DIPSI-2, which can be appended to the beginning of any multi-dimensional NMR sequence that begins on 1H. The TOCSY RF field compels all 1H atoms to behave similarly under the influence of strong coupling and rotating frame cross-relaxation, so that differences in relaxation rates are due primarily to side chain mobility. We apply the scheme to a thermostable mutant Pin1 WW domain and demonstrate that the observed 1H relaxation rates correlate well with two independent NMR measures of side-chain dynamics, cross-correlated 13C relaxation rates in 13CβH2 methylene groups and maximum observable 3J couplings sensitive to the χ1 side chain dihedral angle (3JHα,Hβ, 3JN,Hβ, and 3JCO,Hβ). The most restricted side chains belong to Trp26 and Asn40, which are closely packed to constitute the folding center of the WW domain. None of the other conserved aromatic residues is as immobile as the first tryptophan side chain of the WW domain. The proposed 1H relaxation methodology should make it relatively easy to measure side chain dynamics on uniformly 15N- or 13C-labeled proteins, so long as chemical shift assignments are obtainable.

Core packing of well-defined X-ray and NMR structures is the same.

Numerous studies have investigated the differences and similarities between protein structures determined by solution NMR spectroscopy and those determined by X-ray crystallography. A fundamental question is whether any observed differences are due to differing methodologies or to differences in the behavior of proteins in solution versus in the crystalline state. Here, we compare the properties of the hydrophobic cores of high-resolution protein crystal structures and those in NMR structures, determined using increasing numbers and types of restraints. Prior studies have reported that many NMR structures have denser cores compared with those of high-resolution X-ray crystal structures. Our current work investigates this result in more detail and finds that these NMR structures tend to violate basic features of protein stereochemistry, such as small non-bonded atomic overlaps and few Ramachandran and sidechain dihedral angle outliers. We find that NMR structures solved with more restraints, and which do not significantly violate stereochemistry, have hydrophobic cores that have a similar size and packing fraction as their counterparts determined by X-ray crystallography at high resolution. These results lead us to conclude that, at least regarding the core packing properties, high-quality structures determined by NMR and X-ray crystallography are the same, and the differences reported earlier are most likely a consequence of methodology, rather than fundamental differences between the protein in the two different environments.

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR.

Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods represent an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5’ position, which allows 5’-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.