Solution NMR Experiments Research Articles

Molecular Dynamics Simulation of Ester‐Linked Hen Egg White Lysozyme Reveals the Effect of Missing Backbone Hydrogen Bond Donors on the Protein Structure

AbstractThe three‐dimensional structure of a protein is stabilized by a number of different atomic interactions. One of these is hydrogen bonding. Its influence on the spatial structure of the hen egg white lysozyme is investigated by replacing peptide bonds (except those of the two proline residues) by ester bonds. Molecular dynamics simulations of native and ester‐linked lysozyme are compared with the native crystal structure and with NOE distance bounds derived from solution NMR experiments. The ester‐linked protein shows a slight compaction while losing its native structure. However, it does not unfold completely. The structure remains compact due to its hydrophobic core and a changed network of hydrogen bonds involving side chains.

Absolute Configuration and Docking Study of Canescensterone, a Potent Phytoecdysteroid, with Non‐Lepidopteran Ecdysteroid Receptor Selectivity

AbstractThe rare phytoecdysteroid canescensterone is a potent activator of both natural and engineered ecdysteroid receptor (EcR) gene‐expression systems. This steroid also shows a remarkable selectivity towards non‐lepidopteran compared with lepidopteran EcRs in gene‐switch assays, a property that makes it a novel candidate lead compound for use in insecticide discovery and gene‐switch design. Canescensterone possesses an unusual ecdysteroid structure, including a pyrrole‐2‐carboxylate group attached to C‐24, a moiety not yet found in any other natural ecdysteroid. We have investigated the ambiguous stereochemistry at C‐24 of this steroid by 1D and 2D solution NMR experiments, which resulted in the assignment of the 24R‐configuration and determination of the side‐chain conformation. The completestereochemical assignment allowed the generation of docking models of canescensterone in both a lepidopteran EcR and a non‐lepidopteran EcR, which helps to explain its selective biological activity.

Structure and Dynamics of the Membrane-Bound Form of Pf1 Coat Protein: Implications of Structural Rearrangement for Virus Assembly

The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16–A46) around its long axis changes dramatically (by 160°) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2–G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state 1H/15N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly.

ChemInform Abstract: Low-Temperature Solid-State NMR of Proteins

In surveying the literature of solid-state NMR it is striking that the ratio of reviews to original publications is unusually high. We believe that this reflects on the one hand the evident great potential of this technique for the study of chemical and biological systems, and on the other the formidable technical obstacles to be overcome in obtaining useful results. Despite some reluctance to worsen the review/publication ratio still further, we hope in this article to address, in particular, the community of potential users of solid-state NMR in biology, providing a survey of the area that highlights the practical problems and the means by which they may be tackled, and outlining the results obtained to date within the title field. It is not the intent to provide a comprehensive discussion of the physical hasis of solid-state NMR, which may readily he obtained from other sources,lb but a brief qualitative discussion is included to set the context. Likewise, some selected experimental techniques are described in section IV, but this is not intended to be a complete survey. Reviews of the technique of solid-state NMR can be found elsewhere.?,8 There are also prior reviews of the application of solid-state NMR to biological macromolecules>1° peptides, and proteins.12 In these introductory sections we have in mind in particular those who are quite familiar with NMR experiments in solution, and 90 we try to emphasize both the similarities and the differences between the two states.

Expanded Pyridiniums: Bis‐cyclization of Branched Pyridiniums into Their Fused Polycyclic and Positively Charged Derivatives—Assessing the Impact of Pericondensation on Structural, Electrochemical, Electronic, and Photophysical Features

This study evaluates the impact of the extension of the π-conjugated system of pyridiniums on their various properties. The molecular scaffold of aryl-substituted expanded pyridiniums (referred to as branched species) can be photochemically bis-cyclized into the corresponding fused polycyclic derivatives (referred to as pericondensed species). The representative 1,2,4,6-tetraphenylpyridinium (1(H)) and 1,2,3,5,6-pentaphenyl-4-(p-tolyl)pyridinium (2(Me)) tetra- and hexa-branched pyridiniums are herein compared with their corresponding pericondensed derivatives, the fully fused 9-phenylbenzo[1,2]quinolizino[3,4,5,6-def]phenanthridinium (1(H)f) and the hitherto unknown hemifused 9-methyl-1,2,3-triphenylbenzo[h]phenanthro[9,10,1-def]isoquinolinium (2(Me)f). Combined solid-state X-ray crystallography and solution NMR experiments showed that stacking interactions are barely efficient when the pericondensed pyridiniums are not appropriately substituted. The electrochemical study revealed that the first reduction process of all the expanded pyridiniums occurs at around -1 V vs. SCE, which indicates that the lowest unoccupied molecular orbital (LUMO) remains essentially localized on the pyridinium core regardless of pericondensation. In contrast, the electronic and photophysical properties are significantly affected on going from branched to pericondensed pyridiniums. Typically, the number of absorption bands increases with extended activity towards the visible region (down to ca. 450 nm in MeCN), whereas emission quantum yields are increased by three orders of magnitude (at ca. 0.25 on average). A relationship is established between the observed differential impact of the pericondensation and the importance of the localized LUMO on the properties considered: predominant for the first reduction process compared with secondary for the optical and photophysical properties.

NMR Structural Studies of Membrane Proteins in Lipid Micelles and Lipid Bilayers

Integral membrane proteins regulate major cellular processes in health and disease, including transport, signaling, secretion, adhesion, pathogenesis, and apoptosis, and therefore, represent important targets for structural and functional characterization. Membrane protein structures and functions are regulated by their physical interactions with the surrounding lipids, and NMR is unique in its ability to provide high-resolution information in lipid environments that closely resemble the cellular membranes. Solid-state NMR experiments with proteins in oriented bilayers, and solution NMR experiments with proteins in weakly oriented micelles, provide high-resolution orientation-dependent restraints, which can be combined for protein structure determination and refinement. As previously observed for helical membrane proteins, the NMR spectra of outer membrane barrels in lipid bilayers exhibit characteristic patterns that reflect both protein structure and intra-membrane orientation. Results are presented for mammalian and bacterial α-helical and β-stranded membrane proteins. The NMR structures characterized in lipids provide insights to their distinct functions. (This research was supported by the National Institutes of Health.)

VDAC Studied by Solution NMR: Implications for the Native Structure

The voltage-dependent anion channel (VDAC) is the main pathway for metabolites, small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC has been extensively studied for over thirty years and recently, high-resolution structures of VDAC were determined by X-ray and NMR methods (1-3). These studies used recombinant, refolded protein in membrane mimicking environments and thus the valid question arises how well the resulting atomic structure might resemble the “native” structure of VDAC in the mitochondrial outer membrane (4).Here, we describe implications from solution NMR experiments to this question. Recombinant human VDAC-1 is stably folded in LDAO detergent micelles. Well resolved NMR spectra, including four-dimensional NOESYs, yielded a consistent set of more than 1000 spatial spin-spin correlations that unambiguously define the three-dimensional structure of VDAC-1 (1). The protein forms a 19-stranded beta-barrel with 18 antiparallel and 1 parallel strand pairing. The N-terminal 25 residues are not part of the beta-barrel and solution NMR data link the dynamic properties of this segment to the well-known voltage gating process. The inner diameter of the VDAC-1 barrel is about 25 A, in consistence with published micrographs of native or native-like preparations. The entire outside perimeter of the barrel is hydrophobic and covered by detergent molecules, compatible with a membrane bilayer topology. NMR measurements also revealed interactions of VDAC-1 with beta-NADH and cholesterol, providing a functional connection to experiments on native states of the protein. Furthermore, we can link the micelle-bound state of VDAC structurally and functionally to preparations in phospholipid bilayers by comparing NMR spectra and electron micrographs.(1) Hiller et al. Science 321, 1206 (2008).(2) Bayrhuber et al. PNAS 105, 15370 (2008).(3) Ujwal et al. PNAS 105, 17742 (2008).(4) Colombini. Trends Biochem. Sci. 34, 382 (2009).

Influence of the Solvent on the Self-Assembly of a Modified Amyloid Beta Peptide Fragment. II. NMR and Computer Simulation Investigation

The conformation of a model peptide AAKLVFF based on a fragment of the amyloid beta peptide Abeta16-20, KLVFF, is investigated in methanol and water via solution NMR experiments and molecular dynamics computer simulations. In previous work, we have shown that AAKLVFF forms peptide nanotubes in methanol and twisted fibrils in water. Chemical shift measurements were used to investigate the solubility of the peptide as a function of concentration in methanol and water. This enabled the determination of critical aggregation concentrations. The solubility was lower in water. In dilute solution, diffusion coefficients revealed the presence of intermediate aggregates in concentrated solution, coexisting with NMR-silent larger aggregates, presumed to be beta-sheets. In water, diffusion coefficients did not change appreciably with concentration, indicating the presence mainly of monomers, coexisting with larger aggregates in more concentrated solution. Concentration-dependent chemical shift measurements indicated a folded conformation for the monomers/intermediate aggregates in dilute methanol, with unfolding at higher concentration. In water, an antiparallel arrangement of strands was indicated by certain ROESY peak correlations. The temperature-dependent solubility of AAKLVFF in methanol was well described by a van't Hoff analysis, providing a solubilization enthalpy and entropy. This pointed to the importance of solvophobic interactions in the self-assembly process. Molecular dynamics simulations constrained by NOE values from NMR suggested disordered reverse turn structures for the monomer, with an antiparallel twisted conformation for dimers. To model the beta-sheet structures formed at higher concentration, possible model arrangements of strands into beta-sheets with parallel and antiparallel configurations and different stacking sequences were used as the basis for MD simulations; two particular arrangements of antiparallel beta-sheets were found to be stable, one being linear and twisted and the other twisted in two directions. These structures were used to simulate circular dichroism spectra. The roles of aromatic stacking interactions and charge transfer effects were also examined. Simulated spectra were found to be similar to those observed experimentally (in water or methanol) which show a maximum at 215 or 218 nm due to pi-pi* interactions, when allowance is made for a 15-18 nm red-shift that may be due to light scattering effects.

A Mutagenesis-Free Approach to Assignment of 19F NMR Resonances in Biosynthetically Labeled Proteins

Solution NMR studies of protein structure and dynamics using fluorinated amino acid probes are a valuable addition to the repertoire of existing (13)C, (15)N, and (1)H experiments. Despite the numerous advantages of the (19)F nucleus in NMR, protein studies are complicated by the dependence of resonance assignments on site-directed mutagenesis methods which are laborious and often problematic. Here we report an NMR-based route to the assignment of fluorine resonances in (13)C,(15)N-3-fluoro-l-tyrosine labeled calmodulin. The assignment begins with the correlation of the fluorine nucleus to the delta proton in the novel (13)C,(15)N-enriched probe which is achieved using a CT-HCCF-COSY experiment. Connection to the backbone is made through two additional solution NMR experiments, namely the (H(beta))C(beta)(C(gamma)C(delta))H(delta) and HNCACB. Assignments are completed using either previously published backbone chemical shift data or obtained experimentally provided uniform (13)C,(15)N labeling procedures are employed during protein expression. Additional benefits of the (13)C,(15)N-3-fluoro-l-tyrosine probe include the reduction of spectral overlap through ((13)C(19)F) CT-HSQCs, as well as the ability to monitor side chain dynamics using (19)F T(1), T(2), and the (13)C-(19)F NOE.

Towards thrombosis-targeted zeolitenanoparticles for laser-polarized129Xe MRI

The synthesis and functionalisation of zeolite nanoparticles designed for laser-polarized 129Xe NMR experiments in solution are described. These nanoparticles were functionalized by using original synthesis pathways through semicarbazide COCHO chemistry in order to ensure anchoring of a peptide for targeting biological receptors and attachment of PEG chains for in vivo experiments. Results demonstrated that accessibility of the dissolved noble gas to the micropores was maintained after functionalization. γ Scintigraphy with 111In linked to the zeolite particles has been investigated in order to follow the behaviour of the zeolite nanoparticles in mice. These preliminary 111In scintigraphy experiments showed the localization of the nanoparticles after injection in mice and their biodistribution, as a first proof-of-concept towards in vivo129Xe MRI.

An unambiguous structural elucidation of a 1,3-β- d-glucan obtained from liquid-cultured Grifola frondosa by solution NMR experiments

Grifolan LE (GRN-LE), a purified β- d-glucan, which is obtained from liquid-cultured Grifola frondosa, exhibits various biological activities, including antitumor effects. Significant progress has been made in the study of these effects. However, an unambiguous structural characterization of GRN-LE using NMR spectroscopy has not been carried out as yet. It is well accepted that the biological effects of a β-glucan depend on its primary structure, conformation, and molecular weight. In the present study, we unambiguously elucidate the primary structure of GRN-LE using NMR spectroscopy. The data presented here reveal that GRN-LE comprises a 1,3-β- d-glucan backbone with a single 1,6-β- d-glucosyl side branching unit on every third residue.

Comprehensive Structural Studies of 2′,3′-Difluorinated Nucleosides: Comparison of Theory, Solution, and Solid State

The conformations of three 2',3'-difluoro uridine nucleosides were studied by X-ray crystallography, NMR spectroscopy, and ab initio calculations in an attempt to define the roles that the two vicinal fluorine atoms play in the puckering preferences of the furanose ring. Two of the compounds examined contained fluorine atoms in either the arabino or xylo dispositions at C2' and C3' of a 2',3'-dideoxyuridine system. The third compound also incorporated fluorine atoms in the xylo configuration on the furanose ring but was substituted with a 6-azauracil base in place of uracil. A battery of NMR experiments in D 2O solution was used to identify conformational preferences primarily from coupling constant and NOE data. Both (1)H and (19)F NMR data were used to ascertain the preferred sugar pucker of the furanose ring through the use of the program PSEUROT. Compound-dependent parameters used in the PSEUROT calculations were newly derived from complete sets of conformations calculated from high-level ab initio methods. The solution and theoretical data were compared to the conformations of each molecule in the solid state. It was shown that both gauche and antiperiplanar effects may be operative to maintain a pseudodiaxial arrangement of the C2' and C3' vicinal fluorine atoms. These data, along with previously reported data by us and others concerning monofluorinated nucleoside conformations, were used to propose a model of how fluorine influences different aspects of nucleoside conformations.

NMR Spectroscopy of the Ligand-Binding Core of Ionotropic Glutamate Receptor 2 Bound to 5-Substituted Willardiine Partial Agonists

Glutamate receptors mediate neuronal intercommunication in the central nervous system by coupling extracellular neurotransmitter–receptor interactions to ion channel conductivity. To gain insight into structural and dynamical factors that underlie this coupling, solution NMR experiments were performed on the bilobed ligand-binding core of glutamate receptor 2 in complexes with a set of willardiine partial agonists. These agonists are valuable for studying structure–function relationships because their 5-position substituent size is correlated with ligand efficacy and extent of receptor desensitization, whereas the substituent electronegativity is correlated with ligand potency. NMR results show that the protein backbone amide chemical shift deviations correlate mainly with efficacy and extent of desensitization. Pronounced deviations occur at specific residues in the ligand-binding site and in the two helical segments that join the lobes by a disulfide bond. Experiments detecting conformational exchange show that micro- to millisecond timescale motions also occur near the disulfide bond and vary largely with efficacy and extent of desensitization. These results thus identify regions displaying structural and dynamical dissimilarity arising from differences in ligand–protein interactions and lobe closure that may play a critical role in receptor response. Furthermore, measures of line broadening and conformational exchange for a portion of the ligand-binding site correlate with ligand EC 50 data. These results do not have any correlate in the currently available crystal structures and thus provide a novel view of ligand-binding events that may be associated with agonist potency differences.

Characterization of Enzyme Motions by Solution NMR Relaxation Dispersion

In many enzymes, conformational changes that occur along the reaction coordinate can pose a bottleneck to the rate of conversion of substrates to products. Characterization of these rate-limiting protein motions is essential for obtaining a full understanding of enzyme-catalyzed reactions. Solution NMR experiments such as the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo or off-resonance R 1rho pulse sequences enable quantitation of protein motions in the time range of microseconds to milliseconds. These experiments allow characterization of the conformational exchange rate constant, k ex, the equilibrium populations of the relevant conformations, and the chemical shift differences (Deltaomega) between the conformations. The CPMG experiments were applied to the backbone N-H positions of ribonuclease A (RNase A). To probe the role of dynamic processes in the catalytic cycle of RNase A, stable mimics of the apo enzyme (E), enzyme-substrate (ES) complex, and enzyme-product (EP) complex were formed. The results indicate that the ligand has relatively little influence on the kinetics of motion, which occurs at 1700 s (-1) and is the same as both k cat, and the product dissociation rate constant. Instead, the effect of ligand is to stabilize one of the pre-existing conformations. Thus, these NMR experiments indicate that the conformational change in RNase A is ligand-stabilized and does not appear to be ligand-induced. Further evidence for the coupling of motion and enzyme function comes from the similar solvent deuterium kinetic isotope effect on k ex derived from the NMR measurements and k cat from enzyme kinetic studies. This isotope effect of approximately 2 depends linearly on solvent deuterium content suggesting the involvement of a single proton in RNase A motion and function. Moreover, mutation of His48 to alanine eliminates motion in RNase A and decreases the catalytic turnover rate indicating the involvement of His48, which is far from the active site, in coupling motion and function. For the enzyme triosephosphate isomerase (TIM), the opening and closing motion of a highly conserved active site loop (loop 6) has been implicated in many studies to play an important role in the catalytic cycle of the enzyme. Off-resonance R 1rho experiments were performed on TIM, and results were obtained for amino acid residues in the N-terminal (Val167), and C-terminal (Lys174, Thr177) portions of loop 6. The results indicate that all three loop residues move between the open and closed conformation at about 10,000 s (-1), which is the same as the catalytic rate constant. The O (eta) atom of Tyr208 provides a hydrogen bond to stabilize the closed form of loop 6 by interacting with the amide nitrogen of Ala176; these atoms are outside of hydrogen bonding distance in the open form of the enzyme. Mutation of Tyr208 to phenylalanine results in significant loss of catalytic activity but does not appear to alter the kex value of the N-terminal part of loop 6. Instead, removal of this hydrogen bond appears to result in an increase in the equilibrium population of the open conformer of loop 6, thereby resulting in a loss of activity through a shift in the conformational equilibrium of loop 6. Solution NMR relaxation dispersion experiments are powerful experimental tools that can elucidate protein motions with atomic resolution and can provide insight into the role of these motions in biological function.

A Structural and Theoretical Investigation of Equatorial cis and trans Uranyl Phosphinimine and Uranyl Phosphine Oxide Complexes UO2Cl2(Cy3PNH)2 and UO2Cl2(Cy3PO)2

Phosphinimine ligands (Cy3PNH) readily react with UO2Cl2(THF)3 (THF=tetrahydrofuran) to give UO2Cl2(Cy3PNH)2, which contains strong U-N interactions and exists as cis and trans isomers in the solid and solution state. Solution NMR experiments and computational analysis both support the trans form as the major isomer in solution, although the cis isomer becomes more stabilized with an increase in the dielectric constant of the solvent. Mayer bond orders, energy decomposition analysis, and examination of the molecular orbitals and total electron densities support a more covalent bonding interaction in the U-NHPCy3 bond compared with the analogous bond of the related U-OPCy3 compounds.

NMR Structural Studies of Interactions of a Small, Nonpeptidyl Tpo Mimic with the Thrombopoietin Receptor Extracellular Juxtamembrane and Transmembrane Domains

Thrombopoietin (Tpo) is a glycoprotein growth factor that supports hematopoietic stem cell survival and expansion and is the principal regulator of megakaryocyte growth and differentiation. Several small, nonpeptidyl molecules have been identified as selective human Tpo receptor (hTpoR) agonists. To understand how the small molecule Tpo mimic SB394725 interacts and activates hTpoR, we performed receptor domain swap and mutagenesis studies. The results suggest that SB394725 interacts specifically with the extracellular juxtamembrane region (JMR) and the transmembrane (TM) domain of hTpoR. Solution and solid-state NMR structural studies using a peptide containing the JMR-TM sequences showed that this region of hTpoR, unexpectedly, consists of two alpha-helices separated by a few nonhelical residues. SB394725 interacts specifically with His-499 in the TM domain and a few distinct residues in the JMR-TM region and affects several specific C-terminal TM domain residues. The unique structural information provided by these studies both sheds light on the distinctive mechanism of action of SB394725 and provides valuable insight into the mechanism of ligand-induced cytokine receptor activation.

Fluoro aromatic imine nickel(0) complexes: Synthesis and structural studies

The reaction of [Ni(dippe)] 2(μ-H) 2 with a series of fluoroaromatic imines affords nickel(0) complexes of the type [(dippe)Ni(η 2- C, N)–PhHC NR′Ph], dippe = 1,2-bis(diisopropylphosphine)ethane. Solution NMR experiments as well as X-ray diffraction studies confirmed the π-coordination of the ligand through the C N moiety; the resulting complex found to adopt a distorted tetrahedral geometry around the nickel center. The compounds are thermally stable and decomposition is only observed after long periods of heating above 150 °C.

An Unambiguous Assignment and Structural Analysis Using Solution NMR Experiments of O-Antigen from Escherichia coli ATCC23505 (Serotype O9)

Bacterial lipopolysaccharide from Escherichia coli O9 (O9 LPS) has various characteristic biological activities other than endotoxic activities. The biological activities exhibited depend on the structure of the O-antigen. The O-antigen region of O9 LPS is composed of the mannose homopolysaccharide (MHP). This structure was reported previously, but not all its proton and carbon signals were assigned. In the present study, we completely assign all proton and carbon signals of the O-antigen of O9 LPS using (1)H- and (13)C-NMR spectroscopy, including the DQF-COSY, TOCSY, NOESY, HSQC, H2BC, HSQC-TOCSY and HMBC methods.

O‐DPPB‐Directed Copper‐Mediated and ‐Catalyzed Allylic Substitution with Grignard Reagents

The ortho-diphenylphosphanylbenzoyl (o-DPPB) group was explored as a directing leaving group in copper-mediated and copper-catalyzed allylic substitution with Grignard reagents. Complete control of chemo-, regio- and stereoselectivity with complete syn-1,3-chirality transfer was observed as a result of the directed nature of the reaction. No excess of organometallic reagent is required and the directing group can be recovered quantitatively. Coordination studies in the solid state and in solution have shown that two substrates are bound via the phosphine function of the directing group at copper. Dynamic NMR experiments in solution are in agreement with a ligand-exchange process at copper, a prerequisite for the development of a substoichiometric process.

Site-Specific 13C Chemical Shift Anisotropy Measurements in a Uniformly 15N,13C-Labeled Microcrystalline Protein by 3D Magic-Angle Spinning NMR Spectroscopy

In this Communication, we introduce a 3D magic-angle spinning recoupling experiment that correlates chemical shift anisotropy (CSA) powder line shapes with two dimensions of site-resolved isotropic chemical shifts. The principal tensor elements from 127 ROCSA line shapes are reported, constraining 102 unique backbone and side-chain 13C sites in a microcrystalline protein (the 56 residue beta1 immunoglobulin binding domain of protein G). The tensor elements, determined by fitting to numerical simulations, agree well with quantum chemical predictions. The experiments, therefore, validate calculations of CSAs in a protein of known structure. The data will be useful for the development of side-chain CSA quantum calculations and will aid in the design and interpretation of solution NMR experiments that utilize CSA-dipole cross-correlation to constrain torsion angles or to enhance resolution and sensitivity (such as in TROSY). Furthermore, the methodology described here will enable databases of CSA data to be generated with higher efficiency, for purposes of direct protein structure refinement.