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Hyperpolarized metabolites offer a tremendous sensitivity advantage (>10(4) fold) when measuring flux and enzyme activity in living tissues by magnetic resonance methods. These sensitivity gains can also be applied to mechanistic studies that impose time and metabolite concentration limitations. Here we explore the use of hyperpolarization by dissolution dynamic nuclear polarization (DNP) in mechanistic studies of alanine transaminase (ALT), a well-established biomarker of liver disease and cancer that converts pyruvate to alanine using glutamate as a nitrogen donor. A specific deuterated, (13)C-enriched analog of pyruvic acid, (13)C3D(3)-pyruvic acid, is demonstrated to have advantages in terms of detection by both direct (13)C observation and indirect observation through methyl protons introduced by ALT-catalyzed H-D exchange. Exchange on injecting hyperpolarized (13)C3D(3)-pyruvate into ALT dissolved in buffered (1)H(2)O, combined with an experimental approach to measure proton incorporation, provided information on mechanistic details of transaminase action on a 1.5s timescale. ALT introduced, on average, 0.8 new protons into the methyl group of the alanine produced, indicating the presence of an off-pathway enamine intermediate. The opportunities for exploiting mechanism-dependent molecular signatures as well as indirect detection of hyperpolarized (13)C3-pyruvate and products in imaging applications are discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

While chemical shift prediction has played an important role in aspects of protein NMR that include identification of secondary structure, generation of torsion angle constraints for structure determination, and assignment of resonances in spectra of intrinsically disordered proteins, interest has arisen more recently in using it in alternate assignment strategies for crosspeaks in (1)H-(15)N HSQC spectra of sparsely labeled proteins. One such approach involves correlation of crosspeaks in the spectrum of the native protein with those observed in the spectrum of the denatured protein, followed by assignment of the peaks in the latter spectrum. As in the case of disordered proteins, predicted chemical shifts can aid in these assignments. Some previously developed empirical formulas for chemical shift prediction have depended on basis data sets of 20 pentapeptides. In each case the central residue was varied among the 20 amino common acids, with the flanking residues held constant throughout the given series. However, previous choices of solvent conditions and flanking residues make the parameters in these formulas less than ideal for general application to denatured proteins. Here, we report (1)H and (15)N shifts for a set of alanine based pentapeptides under the low pH urea denaturing conditions that are more appropriate for sparse label assignments. New parameters have been derived and a Perl script was created to facilitate comparison with other parameter sets. A small, but significant, improvement in shift predictions for denatured ubiquitin is demonstrated.

Chemokines are small proteins best known for their role in controlling the migration of diverse cells, particularly leukocytes. Upon binding to their G-protein-coupled receptors on the leukocytes, chemokines stimulate the signaling events that cause cytoskeletal rearrangements involved in cell movement, and migration of the cells along chemokine gradients. Depending on the cell type, chemokines also induce many other types of cellular responses including those related to defense mechanisms, cell proliferation, survival, and development. Historically, most research efforts have focused on the interaction of chemokines with their receptors, where monomeric forms of the ligands are the functionally relevant state. More recently, however, the importance of chemokine interactions with cell surface glycosaminoglycans has come to light, and in most cases appears to involve oligomeric chemokine structures. This review summarizes existing knowledge relating to the structure and function of chemokine oligomers, and emerging methodology for determining structures of complex chemokine assemblies in the future., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The yeast mitochondrial protein Sdh5 is required for the covalent attachment of flavin adenine dinucleotide (FAD) to protein Sdh1, a subunit of the heterotetrameric enzyme succinate dehydrogenase. The NMR structure of Sdh5 represents the first eukaryotic structure of Pfam family PF03937 and reveals a conserved surface region, which likely represents a putative Sdh1-Sdh5 interaction interface. Point mutations in this region result in the loss of covalent flavinylation of Sdh1. Moreover, chemical shift perturbation measurements showed that Sdh5 does not bind FAD in vitro, indicating that it is not a simple cofactor transporter in vivo.

Paramagnetic lanthanide ions when bound to proteins offer great potential for structural investigations that utilize solution nuclear magnetic resonance spectroscopy, magnetic resonance imaging, or optical microscopy. However, many proteins do not have native metal ion binding sites and engineering a chimeric protein to bind an ion while retaining affinity for a protein of interest represents a significant challenge. Here we report the characterization of an immunoglobulin G-binding protein redesigned to include a lanthanide binding motif in place of a loop between two helices (Z-L2LBT). It was shown to bind Tb³⁺ with 130 nM affinity. Ions such as Dy³⁺, Yb³⁺, and Ce³⁺ produce paramagnetic effects on NMR spectra and the utility of these effects is illustrated by their use in determining a structural model of the metal-complexed Z-L2LBT protein and a preliminary characterization of the dynamic distribution of IgG Fc glycan positions. Furthermore, this designed protein is demonstrated to be a novel IgG-binding reagent for magnetic resonance imaging (Z-L2LBT:Gd³⁺ complex) and luminescence microscopy (Z-L2LBT: Tb³⁺ complex)., (Copyright © 2012 The Protein Society.)

The solution nuclear magnetic resonance (NMR) structures and backbone (15)N dynamics of the specialized acyl carrier protein (ACP), RpAcpXL, from Rhodopseudomonas palustris, in both the apo form and holo form modified by covalent attachment of 4'-phosphopantetheine at S37, are virtually identical, monomeric, and correspond to the closed conformation. The structures have an extra α-helix compared to the archetypical ACP from Escherichia coli, which has four helices, resulting in a larger opening to the hydrophobic cavity. Chemical shift differences between apo- and holo-RpAcpXL indicated some differences in the hinge region between α2 and α3 and in the hydrophobic cavity environment, but corresponding changes in nuclear Overhauser effect cross-peak patterns were not detected. In contrast to the NMR structures, apo-RpAcpXL was observed in an open conformation in crystals that diffracted to 2.0 Å resolution, which resulted from movement of α3. On the basis of the crystal structure, the predicted biological assembly is a homodimer. Although the possible biological significance of dimerization is unknown, there is potential that the resulting large shared hydrophobic cavity could accommodate the very long-chain fatty acid (28-30 carbons) that this specialized ACP is known to synthesize and transfer to lipid A. These structures are the first representatives of the AcpXL family and the first to indicate that dimerization may be important for the function of these specialized ACPs.

The terminal carbohydrate residues of the N-glycan on the immunoglobulin G (IgG) fragment crystallizable (Fc) determine whether IgG activates pro- or anti-inflammatory receptors. The IgG Fc alone becomes potently anti-inflammatory upon addition of α2-6-linked N-acetylneuraminic acid residues to the N-glycan, stimulating interest in use of this entity in novel therapies for autoimmune disease [Kaneko et al. (2006) Science313, 670-3]. Complete Fc sialylation has, however, been deemed challenging due to a combination of branch specificity and perceived protection by glycan-protein interactions. Here we report the preparation of high levels of disialylated Fc by using sufficient amounts of a highly active α2-6 sialyltransferase (ST6Gal1) preparation expressed in a transiently transformed human cell culture. Surprisingly, ST6Gal1 sialylated the two termini of the complex-type binantennary glycan in a manner remarkably similar to that observed for the free N-glycan, suggesting the Fc polypeptide does not greatly influence ST6Gal1 specificity. In addition, sialylation of either branch terminus does not appear to dramatically alter the motional behavior of the N-glycan as judged by solution NMR spectroscopy. Together these, data suggest the N-glycan occupies two distinct states: one with both glycan termini sequestered from enzymatic modification by an α1-6Man-branch interaction with the polypeptide surface and the other with both glycan termini exposed to the bulk solvent and free from glycan-polypeptide interactions. The results suggest new modes by which disialylated Fc can act as an anti-inflammatory effector.

Interactions between proteins and glycosaminoglycans (GAGs) of the extracellular matrix are important to the regulation of cellular processes including growth, differentiation and migration. Understanding these processes can benefit greatly from the study of protein-GAG interactions using GAG oligosaccharides of well-defined structure. Materials for such studies have, however, been difficult to obtain because of challenges in synthetic approaches and the extreme structural heterogeneity in GAG polymers. Here, it is demonstrated that diversity in structures of oligosaccharides derived by limited enzymatic digestion of materials from natural sources can be greatly curtailed by a proper selection of combinations of source materials and digestive enzymes, a process aided by an improved understanding of the specificities of certain commercial preparations of hydrolases and lyases. Separation of well-defined oligosaccharides can then be accomplished by size-exclusion chromatography followed by strong anion-exchange chromatography. We focus here on two types of chondroitin sulfate (CS) as starting material (CS-A, and CS-C) and the use of three digestive enzymes with varying specificities (testicular hyaluronidase and bacterial chondroitinases ABC and C). Analysis using nuclear magnetic resonance and mass spectrometry focuses on isolated CS disaccharides and hexasaccharides. In all, 15 CS hexasaccharides have been isolated and characterized. These serve as useful contributions to growing libraries of well-defined GAG oligosaccharides that can be used in further biophysical assays.

CDK2AP1 (cyclin-dependent kinase 2-associated protein 1), corresponding to the gene doc-1 (deleted in oral cancer 1), is a tumor suppressor protein. The doc-1 gene is absent or down-regulated in hamster oral cancer cells and in many other cancer cell types. The ubiquitously expressed CDK2AP1 protein is the only known specific inhibitor of CDK2, making it an important component of cell cycle regulation during G(1)-to-S phase transition. Here, we report the solution structure of CDK2AP1 by combined methods of solution state NMR and amide hydrogen/deuterium exchange measurements with mass spectrometry. The homodimeric structure of CDK2AP1 includes an intrinsically disordered 60-residue N-terminal region and a four-helix bundle dimeric structure with reduced Cys-105 in the C-terminal region. The Cys-105 residues are, however, poised for disulfide bond formation. CDK2AP1 is phosphorylated at a conserved Ser-46 site in the N-terminal "intrinsically disordered" region by IκB kinase ε.

High-quality NMR structures of the homo-dimeric proteins Bvu3908 (69-residues in monomeric unit) from Bacteroides vulgatus and Bt2368 (74-residues) from Bacteroides thetaiotaomicron reveal the presence of winged helix-turn-helix (wHTH) motifs mediating tight complex formation. Such homo-dimer formation by winged HTH motifs is otherwise found only in two DNA-binding proteins with known structure: the C-terminal wHTH domain of transcriptional activator FadR from E. coli and protein TubR from B. thurigensis, which is involved in plasmid DNA segregation. However, the relative orientation of the wHTH motifs is different and residues involved in DNA-binding are not conserved in Bvu3908 and Bt2368. Hence, the proteins of the present study are not very likely to bind DNA, but are likely to exhibit a function that has thus far not been ascribed to homo-dimers formed by winged HTH motifs. The structures of Bvu3908 and Bt2368 are the first atomic resolution structures for PFAM family PF10771, a family of unknown function (DUF2582) currently containing 128 members.

Obesity and age are risk factors for feline diabetes. This study aimed to test the hypothesis that age, long-term obesity, and dietary composition would lead to peripheral and hepatorenal insulin resistance, indicated by higher endogenous glucose production (EGP) in the fasted and postprandial state, higher blood glucose and insulin, and higher leptin, free thyroxine, and lower adiponectin concentrations. Using triple tracer-(2)H(2)O, [U-(13)C(3)] propionate, and [3,4-(13)C(2)] glucose infusion, and indirect calorimetry-we investigated carbohydrate and fat metabolic pathways in overnight-fasted neutered cats (13 young lean, 12 old lean, and 12 old obese), each fed three different diets (high protein with and without polyunsaturated fatty acids, and high carbohydrate) in a crossover design. EGP was lowest in fasted and postprandial obese cats despite peripheral insulin resistance, indicated by hyperinsulinemia. Gluconeogenesis was the most important pathway for EGP in all groups, but glycogen contributed significantly. Insulin and leptin concentrations were higher in old than in young lean cats; adiponectin was lowest in obese cats but surprisingly highest in lean old cats. Diet had little effect on metabolic parameters. We conclude that hepatorenal insulin resistance does not develop in the fasted or postprandial state, even in long-term obese cats, allowing the maintenance of euglycemia through lowering EGP. Glycogen plays a major role in EGP, especially in lean fasted cats, and in the postprandial state. Aging may predispose to insulin resistance, which is a risk factor for diabetes in cats. Mechanisms underlying the high adiponectin of healthy old lean cats need to be further explored.

Hyperpolarization greatly enhances opportunities to observe in vivo metabolic processes in real time. Accessible timescales are, however, limited by nuclear spin relaxation times, and sensitivity is limited by magnetogyric ratios of observed nuclei. The majority of applications to date have involved direct (13)C observation of metabolites with non-protonated carbons at sites of interest ((13)C enriched carbonyls, for example), a choice that extends relaxation times and yields moderate sensitivity. Interest in (15)N containing metabolites is equally high but non-protonated sites are rare and direct (15)N observation insensitive. Here an approach is demonstrated that extends applications to protonated (15)N sites with high sensitivity. The normally short relaxation times are lengthened by initially replacing protons (H) with deuterons (D) and low sensitivity detection of (15)N is avoided by indirect detection through protons reintroduced by H/D exchange. A pulse sequence is presented that periodically samples (15)N polarization at newly protonated sites by INEPT transfer to protons while returning (15)N magnetization of deuterated sites to the +Z axis to preserve polarization for subsequent samplings. Applications to (15)ND(2)-amido-glutamine are chosen for illustration. Glutamine is an important regulator and a direct donor of nitrogen in cellular metabolism. Potential application to in vivo observation is discussed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Most multi-dimensional solution NMR experiments connect one dimension to another using coherence transfer steps that involve evolution under scalar couplings. While experiments of this type have been a boon to biomolecular NMR the need to work on ever larger systems pushes the limits of these procedures. Spin relaxation during transfer periods for even the most efficient (15)N-(1)H HSQC experiments can result in more than an order of magnitude loss in sensitivity for molecules in the 100 kDa range. A relatively unexploited approach to preventing signal loss is to avoid coherence transfer steps entirely. Here we describe a scheme for multi-dimensional NMR spectroscopy that relies on direct frequency encoding of a second dimension by multi-frequency decoupling during acquisition, a technique that we call MD-DIRECT. A substantial improvement in sensitivity of (15)N-(1)H correlation spectra is illustrated with application to the 21 kDa ADP ribosylation factor (ARF) labeled with (15)N in all alanine residues. Operation at 4°C mimics observation of a 50 kDa protein at 35°C., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Few solution NMR pulse sequences exist that are explicitly designed to characterize carbohydrates (glycans). This is despite the essential role carbohydrate motifs play in cell-cell communication, microbial pathogenesis, autoimmune disease progression and cancer metastasis, and despite that fact that glycans, often shed to extra-cellular fluids, can be diagnostic of disease. Here we present a suite of two dimensional coherence experiments to measure three different correlations (H3-C2, H3-C1, and C1-C2) on sialic acids, a group of nine-carbon carbohydrates found on eukaryotic cell surfaces that often play a key role in disease processes. The chemical shifts of the H3, C2, and C1 nuclei of sialic acids are sensitive to carbohydrate linkage, linkage conformation, and ionization state of the C1 carboxylate. The experiments reported include rigorous filter elements to enable detection and characterization of isotopically labeled sialic acids with high sensitivity in living cells and crude isolates with minimal interference from unwanted signals arising from the ~1% (13)C-natural abundance of cellular metabolites. Application is illustrated with detection of sialic acids on living cells, in unpurified mixtures, and at the terminus of the N-glycan on the 55 kDa immunoglobulin G Fc.

CCL5 (RANTES) is a proinflammatory chemokine known to activate leukocytes through its receptor, CCR5. Although the monomeric form of CCL5 is sufficient to cause cell migration in vitro, CCL5's propensity for aggregation is essential for migration in vivo, T cell activation and apoptosis, and HIV entry into cells. However, there is currently no structural information on CCL5 oligomers larger than the canonical CC chemokine dimer. In this study the solution structure of a CCL5 oligomer was investigated using an integrated approach, including NMR residual dipolar couplings to determine allowed relative orientations of the component monomers, SAXS to restrict overall shape, and hydroxyl radical footprinting and NMR cross-saturation experiments to identify interface residues. The resulting model of the CCL5 oligomer provides a basis for explaining the disaggregating effect of E66 and E26 mutations and suggests mechanisms by which glycosaminoglycan binding may promote oligomer formation and facilitate cell migration in vivo., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Nuclear magnetic resonance paramagnetic relaxation enhancement (PRE) measures long-range distances to isotopically labeled residues, providing useful constraints for protein structure prediction. The method usually requires labor-intensive conjugation of nitroxide labels to multiple locations on the protein, one at a time. Here a computational procedure, based on protein sequence and simple secondary structure models, is presented to facilitate optimal placement of a minimum number of labels needed to determine the correct topology of a helical transmembrane protein. Tests on DsbB (four helices) using just one label lead to correct topology predictions in four of five cases, with the predicted structures <6 Å to the native structure. Benchmark results using simulated PRE data show that we can generally predict the correct topology for five and six to seven helices using two and three labels, respectively, with an average success rate of 76% and structures of similar precision. The results show promise in facilitating experimentally constrained structure prediction of membrane proteins., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

GmACP3 from Geobacter metallireducens is a specialized acyl carrier protein (ACP) whose gene, gmet&#95;2339, is located near genes encoding many proteins involved in lipopolysaccharide (LPS) biosynthesis, indicating a likely function for GmACP3 in LPS production. By overexpression in Escherichia coli, about 50% holo-GmACP3 and 50% apo-GmACP3 were obtained. Apo-GmACP3 exhibited slow precipitation and non-monomeric behavior by (15)N NMR relaxation measurements. Addition of 4'-phosphopantetheine (4'-PP) via enzymatic conversion by E. coli holo-ACP synthase resulted in stable >95% holo-GmACP3 that was characterized as monomeric by (15)N relaxation measurements and had no indication of conformational exchange. We have determined a high-resolution solution structure of holo-GmACP3 by standard NMR methods, including refinement with two sets of NH residual dipolar couplings, allowing for a detailed structural analysis of the interactions between 4'-PP and GmACP3. Whereas the overall four helix bundle topology is similar to previously solved ACP structures, this structure has unique characteristics, including an ordered 4'-PP conformation that places the thiol at the entrance to a central hydrophobic cavity near a conserved hydrogen-bonded Trp-His pair. These residues are part of a conserved WDSLxH/N motif found in GmACP3 and its orthologs. The helix locations and the large hydrophobic cavity are more similar to medium- and long-chain acyl-ACPs than to other apo- and holo-ACP structures. Taken together, structural characterization along with bioinformatic analysis of nearby genes suggests that GmACP3 is involved in lipid A acylation, possibly by atypical long-chain hydroxy fatty acids, and potentially is involved in synthesis of secondary metabolites.

Solving structures of native oligomeric protein complexes using traditional high-resolution NMR techniques remains challenging. However, increased utilization of computational platforms, and integration of information from less traditional NMR techniques with data from other complementary biophysical methods, promises to extend the boundary of NMR-applicable targets. This article reviews several of the techniques capable of providing less traditional and complementary structural information. In particular, the use of orientational constraints coming from residual dipolar couplings and residual chemical shift anisotropy offsets are shown to simplify the construction of models for oligomeric complexes, especially in cases of weak homo-dimers. Combining this orientational information with interaction site information supplied by computation, chemical shift perturbation, paramagnetic surface perturbation, cross-saturation and mass spectrometry allows high resolution models of the complexes to be constructed with relative ease. Non-NMR techniques, such as mass spectrometry, EPR and small angle X-ray scattering, are also expected to play increasingly important roles by offering alternative methods of probing the overall shape of the complex. Computational platforms capable of integrating information from multiple sources in the modeling process are also discussed in the article. And finally a new, detailed example on the determination of a chemokine tetramer structure will be used to illustrate how a non-traditional approach to oligomeric structure determination works in practice., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The N-glycan at Asn297 of the immunoglobulin G Fc fragment modulates cellular responses of the adaptive immune system. However, the underlying mechanism remains undefined, as existing structural data suggest the glycan does not directly engage cell surface receptors. Here we characterize the dynamics of the glycan termini using solution NMR spectroscopy. Contrary to previous conclusions based on X-ray crystallography and limited NMR data, our spin relaxation studies indicate that the termini of both glycan branches are highly dynamic and experience considerable motion in addition to tumbling of the Fc molecule. Relaxation dispersion and temperature-dependent chemical shift perturbations demonstrate exchange of the α1-6Man-linked branch between a protein-bound and a previously unobserved unbound state, suggesting the glycan samples conformational states that can be accessed by glycan-modifying enzymes and possibly glycan recognition domains. These findings suggest a role for Fc-glycan dynamics in Fc-receptor interactions and enzymatic glycan remodeling.

YbbR domains are widespread throughout Eubacteria and are expressed as monomeric units, linked in tandem repeats or cotranslated with other domains. Although the precise role of these domains remains undefined, the location of the multiple YbbR domain-encoding ybbR gene in the Bacillus subtilis glmM operon and its previous identification as a substrate for a surfactin-type phosphopantetheinyl transferase suggests a role in cell growth, division, and virulence. To further characterize the YbbR domains, structures of two of the four domains (I and IV) from the YbbR-like protein of Desulfitobacterium hafniense Y51 were solved by solution nuclear magnetic resonance and X-ray crystallography. The structures show the domains to have nearly identical topologies despite a low amino acid identity (23%). The topology is dominated by β-strands, roughly following a "figure 8" pattern with some strands coiling around the domain perimeter and others crossing the center. A similar topology is found in the C-terminal domain of two stress-responsive bacterial ribosomal proteins, TL5 and L25. Based on these models, a structurally guided amino acid alignment identifies features of the YbbR domains that are not evident from naïve amino acid sequence alignments. A structurally conserved cis-proline (cis-Pro) residue was identified in both domains, though the local structure in the immediate vicinities surrounding this residue differed between the two models. The conservation and location of this cis-Pro, plus anchoring Val residues, suggest this motif may be significant to protein function., (Copyright © 2011 The Protein Society.)

The surfaces of mammalian cells are coated with complex carbohydrates, many terminated with a negatively charged N-acetylneuraminic acid residue. This motif is specifically targeted by pathogens, including influenza viruses and many pathogenic bacteria, to gain entry into the cell. A necessary step in the influenza virus life cycle is the release of viral particles from the cell surface; this is achieved by cleaving N-acetylneuraminic acid from cell surface glycans with a virally-produced neuraminidase. Here we present a laboratory exercise to model this process using a glycoprotein as a glycan carrier and using real time nuclear magnetic resonance (NMR) spectroscopy to monitor N-acetylneuraminic acid release as catalyzed by neuraminidase. A time-resolved two dimensional data processing technique, statistical total correlation spectroscopy (STOCSY), enhances the resolution of the complicated 1D glycoprotein spectrum and isolates characteristic peaks corresponding to substrates and products. This exercise is relatively straightforward and leads students through a wide range of biologically and chemically relevant procedures, including use of NMR spectroscopy, enzymology and data processing techniques.

The use of nondestructive NMR spectroscopy for enzymatic studies offers unique opportunities to identify nearly all enzymatic byproducts and detect unstable short-lived products or intermediates at the molecular level; however, numerous challenges must be overcome before it can become a widely used tool. The biosynthesis of acetyl-coenzyme A (acetyl-CoA) by acetyl-CoA synthetase is used here as a case study for the development of an analytical NMR-based time-course assay platform. We describe an algorithm to deconvolve superimposed spectra into spectra for individual molecules, and further develop a model to simulate the acetyl-CoA synthetase enzyme reaction network using the data derived from time-course NMR. Simulation shows indirectly that synthesis of acetyl-CoA is mediated via an enzyme-bound intermediate (possibly acetyl-AMP) and is accompanied by a nonproductive loss from an intermediate. The ability to predict enzyme function based on partial knowledge of the enzymatic pathway topology is also discussed., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The traditional NMR-based method for determining oligomeric protein structure usually involves distinguishing and assigning intra- and intersubunit NOEs. This task becomes challenging when determining symmetric homo-dimer structures because NOE cross-peaks from a given pair of protons occur at the same position whether intra- or intersubunit in origin. While there are isotope-filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (K(d) 3.4 ± 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape-based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed., (Copyright © 2010 The Protein Society.)

Residual dipolar coupling (RDC) and residual chemical shift anisotropy (RCSA) report on orientational properties of a dipolar bond vector and a chemical shift anisotropy principal axis system, respectively. They can be highly complementary in the analysis of backbone structure and dynamics in proteins as RCSAs generally include a report on vectors out of a peptide plane while RDCs usually report on in-plane vectors. Both RDC and RCSA average to zero in isotropic solutions and require partial orientation in a magnetic field to become observable. While the alignment and measurement of RDC has become routine, that of RCSA is less common. This is partly due to difficulties in providing a suitable isotopic reference spectrum for the measurement of the small chemical shift offsets coming from RCSA. Here we introduce a device (modified NMR tube) specifically designed for accurate measurement of reference and aligned spectra for RCSA measurements, but with a capacity for RDC measurements as well. Applications to both soluble and membrane anchored proteins are illustrated.

ADP ribosylation factors (Arfs) are N-myristoylated GTP/GDP switch proteins that have key regulatory roles in vesicle transport in eukaryotic cells. ARFs execute their roles by anchoring to membrane surfaces, where they interact with other proteins to initiate budding and maturation of transport vesicles. However, existing structures of Arf*GTP are limited to nonmyristoylated and truncated forms with impaired membrane binding. We report a high-resolution NMR structure for full-length myristoylated yeast (Saccharomyces cerevisiae) Arf1 in complex with a membrane mimic. The two-domain structure, in which the myristoylated N-terminal helix is separated from the C-terminal domain by a flexible linker, suggests a level of adaptability in binding modes for the myriad of proteins with which Arf interacts and allows predictions of specific lipid binding sites on some of these proteins.

Characterization of glycosaminoglycans (GAGs), including chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS), is important in developing an understanding of cellular function and in assuring quality of preparations destined for biomedical applications. While use of (1)H and (13)C NMR spectroscopy has become common in characterization of these materials, spectra are complex and difficult to interpret when a more heterogeneous GAG type or a mixture of several types is present. Herein a method based on (1)H-(15)N two-dimensional NMR experiments is described. The (15)N- and (1)H-chemical shifts of amide signals from (15)N-containing acetylgalactosamines in CSs are shown to be quite sensitive to the sites of sulfation (4-, 6-, or 4,6-) and easily distinguishable from those of DS. The amide signals from residual (15)N-containing acetylglucosamines in HS are shown to be diagnostic of the presence of these GAG components as well. Most data were collected at natural abundance of (15)N despite its low percentage. However enrichment of the (15)N-content in GAGs using metabolic incorporation from (15)N-glutamine added to cell culture media is also demonstrated and used to distinguish metabolic states in different cell types.

Long range interactions between nuclear spins and paramagnetic ions can serve as a sensitive monitor of internal motion of various parts of proteins, including functional loops and separate domains. In the case of interdomain motion, the interactions between the ion and NMR-observable nuclei are modulated in direction and magnitude mainly by a combination of overall and interdomain motions. The effects on observable parameters such as paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS) can, in principle, be used to characterize motion. These parameters are frequently used for the purpose of structural refinements. However, their use to probe actual domain motions is less common and is lacking a proper theoretical treatment from a motional perspective. In this work, a suitable spin Hamiltonian is incorporated in a two body diffusion model to produce the time correlation function for the nuclear spin-paramagnetic ion interactions. Simulated observables for nuclei in different positions with respect to the paramagnetic ion are produced. Based on these simulations, it demonstrated that both the PRE and the PCS can be very sensitive probes of domain motion. Results for different nuclei within the protein sense different aspects of the motions. Some are more sensitive to the amplitude of the internal motion, others are more sensitive to overall diffusion rates, allowing separation of these contributions. Experimentally, the interaction strength can also be tuned by substitution of different paramagnetic ions or by varying magnetic field strength (in the case of lanthanides) to allow the use of more detailed diffusion models without reducing the reliability of data fitting.

Challenges for structural characterization of membrane-bound glycosphingolipids include their high internal dynamic motions and their physical proximity to membrane surfaces. Here we demonstrate that NMR paramagnetic relaxation enhancement can be used, alongside independent molecular dynamics simulations and an outer-sphere relaxation model, to quantitatively characterize the presentation (insertion depth and orientation relative to a membrane surface) of ganglioside GM1 in biologically relevant membrane environments. Longitudinal and transverse paramagnetic relaxation enhancement effects were measured for GM1, anchored to phospholipid bicelles, using both water-soluble and membrane-anchored paramagnetic probes, respectively. A method was developed to rapidly calculate paramagnetic relaxation enhancement effects from thousands of structures taken from a simulation of GM1 in a phospholipid bilayer. The combined computational and experimental approach yielded experimentally verified atomic-resolution 3D models of a highly plastic membrane-bound biomolecule.

The alpha-2,6-sialyltransferase (ST6Gal-I) is a key enzyme that regulates the distribution of sialic acid-containing molecules on mammalian cell surfaces. However, the fact that its native form is membrane-bound and glycosylated has made structural characterization by X-ray crystallography of this eukaryotic protein difficult. Its large size ( approximately 40 kDa for just the catalytic domain) also poses a challenge for complete structure determination by nuclear magnetic resonance (NMR). However, even without complete structure determination, there are NMR strategies that can return targeted information about select regions of the protein, including information about the active site as seen from the perspective of its bound ligands. Here, in a continuation of a previous study, a spin-labeled mimic of a glycan acceptor ligand is used to identify additional amino acids located in the protein active site. In addition, the spin-labeled donor is used to characterize the relative placement of the two bound ligands. The ligand conformation and protein-ligand contact surfaces are studied by transferred nuclear Overhauser effects (trNOEs) and saturation transfer difference (STD) experiments. The data afforded by the methods mentioned above lead to a geometric model of the bound substrates that in many ways carries an imprint of the ST6Gal-I binding site.

Sialylated forms of the Fc fragment of immunoglobulin G, produced by the human alpha2-6 sialyltransferase ST6Gal-I, were identified as potent anti-inflammatory mediators in a mouse model of rheumatoid arthritis and are potentially the active components in intravenous IgG anti-inflammatory therapies. The activities and specificities of hST6Gal-I are, however, poorly characterized. Here MS and NMR methodology demonstrates glycan modification occurs in a branch-specific manner with the alpha1-3Man branch of the complex, biantennary Fc glycan preferentially sialylated. Interestingly, this substrate preference is preserved when using a released glycan, suggesting that the apparent occlusion of glycan termini in Fc crystal structures does not dominate specificity.

Residual dipolar couplings (RDCs) between NC' and NC(alpha) atoms in polypeptide backbones of proteins contain information on the orientation of bond vectors that is complementary to that contained in NH RDCs. The (1)J(NC)(alpha) and (2)J(NC)(alpha) scalar couplings between these atoms also display a Karplus relation with the backbone torsion angles and report on secondary structure. However, these N-C couplings tend to be small and they are frequently unresolvable in frequency domain spectra having the broad lines characteristic of large proteins. Here a TROSY-based J-modulated approach for the measurement of small (15)N-(13)C couplings in large proteins is described. The cross-correlation interference effects inherent in TROSY methods improve resolution and signal to noise ratios for large proteins, and the use of J-modulation to encode couplings eliminates the need to remove frequency distortions from overlapping peaks during data analysis. The utility of the method is demonstrated by measurement of (1)J(NC'), (1)J(NC)(alpha) , and (2)J(NC)(alpha) scalar couplings and (1)D(NC') and D(NC)(alpha) residual dipolar couplings for the myristoylated yeast ARF1.GTPgammas protein bound to small lipid bicelles, a system with an effective molecule weight of approximately 70kDa.

Protein NMR assignments of large proteins using traditional triple resonance techniques depends on double or triple labeling of samples with (15)N, (13)C, and (2)H. This is not always practical with proteins that require expression in nonbacterial hosts. Labeling with isotopically labeled versions of single amino acids (sparse labeling) often is possible; however, resonance assignment then requires a new strategy. Here a procedure for the assignment of cross-peaks in (15)N-(1)H correlation spectra of sparsely labeled proteins is presented. It relies on the correlation of proton-deuterium amide exchange rates in native and denatured spectra of the intact protein, followed by correlation of chemical shifts in the spectra of the denatured protein with chemical shifts of sequenced peptides derived from the protein. The procedure is successfully demonstrated on a sample of a protein, Galectin-3, selectively labeled with (15)N at all alanine residues.

This Perspective, arising from a workshop held in July 2008 in Buffalo NY, provides an overview of the role NMR has played in the United States Protein Structure Initiative (PSI), and a vision of how NMR will contribute to the forthcoming PSI-Biology program. NMR has contributed in key ways to structure production by the PSI, and new methods have been developed which are impacting the broader protein NMR community.

Obesity is a risk factor for type 2 diabetes in cats. The risk of developing diabetes is severalfold greater for male cats than for females, even after having been neutered early in life. The purpose of this study was to investigate the role of different metabolic pathways in the regulation of endogenous glucose production (EGP) during the fasted state considering these risk factors. A triple tracer protocol using (2)H(2)O, [U-(13)C(3)]propionate, and [3,4-(13)C(2)]glucose was applied in overnight-fasted cats (12 lean and 12 obese; equal sex distribution) fed three different diets. Compared with lean cats, obese cats had higher insulin (P < 0.001) but similar blood glucose concentrations. EGP was lower in obese cats (P < 0.001) due to lower glycogenolysis and gluconeogenesis (GNG; P < 0.03). Insulin, body mass index, and girth correlated negatively with EGP (P < 0.003). Female obese cats had approximately 1.5 times higher fluxes through phosphoenolpyruvate carboxykinase (P < 0.02) and citrate synthase (P < 0.05) than male obese cats. However, GNG was not higher because pyruvate cycling was increased 1.5-fold (P < 0.03). These results support the notion that fasted obese cats have lower hepatic EGP compared with lean cats and are still capable of maintaining fasting euglycemia, despite the well-documented existence of peripheral insulin resistance in obese cats. Our data further suggest that sex-related differences exist in the regulation of hepatic glucose metabolism in obese cats, suggesting that pyruvate cycling acts as a controlling mechanism to modulate EGP. Increased pyruvate cycling could therefore be an important factor in modulating the diabetes risk in female cats.

ADP-ribosylation factors (ARFs) are small (21 kDa), monomeric GTPases that are important regulators of membrane traffic. When membrane bound, they recruit soluble adaptors to membranes and trigger the assembly of coating complexes involved in cargo selection and vesicular budding. N-myristoylation is a conserved feature of all ARF proteins that is required for its biological functions, although the mechanism(s) by which the myristate acts in ARF functions is not fully understood. Here we present the structure of a myristoylated ARF1 protein, determined by solution NMR methods, and an assessment of the influence of myristoylation on association of ARF1.GDP and ARF1.GTP with lipid bilayers. A model in which myristoylation contributes to both the regulation of guanine nucleotide exchange and stable membrane association is supported.

The structure of the 142-residue protein Q8ZP25&#95;SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE&#95;ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247-D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the 'thioredoxin-like clan'. However, protein HYAE&#95;ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE&#95;ECOLI was previously classified as a [NiFe] hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.

Glycans that are either N-linked to asparagine or O-linked to serine or threonine are the hallmark of glycoproteins, a class of protein that dominates the mammalian proteome. These glycans perform important functions in cells and in some cases are required for protein activity. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for studying glycan structure and interactions, particularly in a form that exploits heteronuclei such as 13C. Here an approach is presented that that uses alpha-2,6-sialyltransferase (ST6Gal-I) to enzymatically add 13C-N-acetylneuraminic acid (NeuAc or sialic acid) to glycoproteins after their preparation using nonbacterial hosts. ST6Gal-I is itself a glycoprotein, and in this initial application, labeling of its own glycans and observation of these glycans by NMR are illustrated. The catalytic domain from rat ST6Gal-I was expressed in mammalian HEK293 cells. The glycans from the two glycosylation sites were analyzed with mass spectrometry and found to contain sialylated biantennary structures. The isotopic labeling approach involved removal of the native NeuAc residues from ST6Gal-I with neuraminidase, separation of the neuramindase with a lectin affinity column, and addition of synthesized 13C-CMP-NeuAc to the desialylated ST6Gal-I. Chemical shift dispersion due to the various 13C-NeuAc adducts on ST6Gal-I was observed in a 3D experiment correlating 1H-13C3-13C2 atoms of the sugar ring.

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.

The determination of the location and conformation of a natural ligand bound to a protein receptor is often a first step in the rational design of molecules that can modulate receptor function. NMR observables, including NOEs, often provide the basis for these determinations. However, when ligands are carbohydrates, interactions mediated by extensive hydrogen-bonding networks often reduce or eliminate NOEs between ligand and protein protons. In these cases, it is useful to look to other distance- and orientation-dependent observables that can constrain the geometry of ligand-protein complexes. Here we illustrate the use of paramagnetism-based NMR constraints, including pseudo-contact shifts (PCS) and field-induced residual dipolar couplings (RDCs). When a paramagnetic center can be attached to the protein, field-induced RDCs and PCS reflect only bound-state properties of the ligand, even when averages over small fractions of bound states and large fractions of free states are observed. The effects can also be observed over a long range, making it possible to attach a paramagnetic center to a remote part of the protein. The system studied here is a Galectin-3-lactose complex. A lanthanide-binding peptide showing minimal flexibility with respect to the protein was integrated into the C terminus of an expression construct for the Galectin-3-carbohydrate-binding domain. Dysprosium ion, which has a large magnetic susceptibility anisotropy, was complexed to the peptide, making it possible to observe both PCSs and field-induced RDCs for the protein and the ligand. The structure determined from these constraints shows agreement with a crystal structure of a Galectin-3-N-acetyllactosamine complex.

Relaxation rates in NMR are usually measured by intensity modulation as a function of a relaxation delay during which the relaxation mechanism of interest is effective. Other mechanisms are often suppressed during the relaxation delay by pulse sequences which eliminate their effects, or cancel their effects when two data sets with appropriate combinations of relaxation rate effects are added. Cross-correlated relaxation (CCR) involving dipole-dipole and CSA interactions differ from auto-correlated relaxation (ACR) in that the signs of contributions can be changed by inverting the state of one spin involved in the dipole-dipole interaction. This property has been exploited previously using CPMG sequences to refocus CCR while ACR evolves. Here we report a new pulse scheme that instead eliminates intensity modulation by ACR and thus allows direct measurement of CCR. The sequence uses a constant time relaxation period for which the contribution of ACR does not change. An inversion pulse is applied at various points in the sequence to effect a decay that depends on CCR only. A 2-D experiment is also described in which chemical shift evolution in the indirect dimension can share the same constant period. This improves sensitivity by avoiding the addition of a separate indirect dimension acquisition time. We illustrate the measurement of residue specific CCR rates on the non-myristoylated yeast ARF1 protein and compare the results to those obtained following the conventional method of measuring the decay rates of the slow and fast-relaxing (15)N doublets. The performances of the two methods are also quantitatively evaluated by simulation. The analysis shows that the shared constant-time CCR (SCT-CCR) method significantly improves sensitivity.

The solution structure of Alg13, the glycosyl donor-binding domain of an important bipartite glycosyltransferase in the yeast Saccharomyces cerevisiae, is presented. This glycosyltransferase is unusual in that it is active only in the presence of a binding partner, Alg14. Alg13 is found to adopt a unique topology among glycosyltransferases. Rather than the conventional Rossmann fold found in all GT-B enzymes, the N-terminal half of the protein is a Rossmann-like fold with a mixed parallel and antiparallel beta sheet. The Rossmann fold of the C-terminal half of Alg13 is conserved. However, although conventional GT-B enzymes usually possess three helices at the C terminus, only two helices are present in Alg13. Titration of Alg13 with both UDP-GlcNAc, the native glycosyl donor, and a paramagnetic mimic, UDP-TEMPO, shows that the interaction of Alg13 with the sugar donor is primarily through the residues in the C-terminal half of the protein.

Protein oligomerization serves an important function in biological processes, yet solving structures of protein oligomers has always been a challenge. For solution NMR, the challenge arises both from the increased size of these systems and, in the case of homo-oligomers, from ambiguities in assignment of intra- as opposed to intersubunit NOEs. In this study, we present a residual dipolar coupling (RDC)-assisted method for constructing models of homo-oligomers with purely rotational symmetry. Utilizing the fact that one of the principal axes of the tensor describing the alignment needed for RDC measurement is always parallel to the oligomer symmetry axis, it is possible to greatly restrict possible models for the oligomer. Here, it is shown that, if the monomer structure is known, all allowed dimer models can be constructed using a grid search algorithm and evaluated based on RDC simulations and the quality of the interface between the subunits. Using the Bacillus subtilis protein YkuJ as an example, it is shown that the evaluation criteria based on just two sets of NH RDCs are very selective and can unambiguously produce a model in good agreement with an existing X-ray structure of YkuJ.

A method of identifying the best structural model for a protein of unknown structure from a list of structural candidates using unassigned 15N1H residual dipolar coupling (RDC) data and probability density profile analysis (PDPA) is described. Ten candidate structures have been obtained for the structural genomics target protein PF2048.1 using ROBETTA. 15N1H residual dipolar couplings have been measured from NMR spectra of the protein in two alignment media and these data have been analyzed using PDPA to rank the models in terms of their ability to represent the actual structure. A number of advantages in using this method to characterize a protein structure become apparent. RDCs can easily and rapidly be acquired, and without the need for assignment, the cost and duration of data acquisition is greatly reduced. The approach is quite robust with respect to imprecise and missing data. In the case of PF2048.1, a 79 residue protein, only 58 and 55 of the total RDC data were observed. The method can accelerate structure determination at higher resolution using traditional NMR spectroscopy by providing a starting point for the addition of NOEs and other NMR structural data.

REDCRAFT, a new open source software tool that accommodates the analysis of RDC data for simultaneous structure and dynamics characterization of proteins is presented in this article. Simultaneous consideration of structure and motion is believed to be necessary for accurate representation of the solution-state of a protein. REDCRAFT is designed to primarily utilize RDC data from multiple alignment media in two stages. During Stage-I, a list of possible torsion angles joining any two neighboring peptide planes is ranked based on their fitness to experimental constraints; in Stage-II, a dipeptide fragment is extended by addition of one peptide plane at a time. The algorithm adopted by REDCRAFT is very efficient and can produce a structure for an 80 residue protein within two hours on a typical desktop computer. REDCRAFT exhibits robustness with respect to noise and missing data. REDCRAFT describes the overall alignment of the molecule in the form of an order tensor matrix and is capable of identifying peptide fragments with internal dynamics. Identification of the location of internal motion will permit a more accurate structural representation. Experimental data from two proteins as well as simulated data are presented to illustrate the capabilities of REDCRAFT in both structure determination and identification of the dynamical regions.

A new method is presented for the retrieval of information on the conformation of glycosaminoglycan oligomers in solution. The method relies on the replacement of acetyl groups in isolated native oligomers with 13C labeled acetyl groups and the extraction of orientational constraints from residual dipolar couplings (RDCs) and chemical shift anisotropy (CSA) offsets observed in NMR spectra of partially oriented samples. A novel method for assignment of resonances based on the correlation of resonance intensities with isotope ratios determined from mass spectrometric analysis is also presented. The combined methods are used in conjunction with more traditional NMR structural data to determine the solution structure of a pentasaccharide, GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc4S(beta1-4)GlcA(beta1-3)GalNAc4S-ol, derived by enzymatic hydrolysis of chondroitin sulfate. The geometry derived is compared to that for similar molecules that have been reported in the literature, and prospects for use of the new types of data in the study of protein-bound oligosaccharides are discussed.

We present a nuclear magnetic resonance (NMR) study in solution of the structures of human normal hemoglobin (Hb A) in the deoxy or unligated form in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using 15N-1H residual dipolar coupling (RDC) measurements. There are several published crystal structures for deoxyhemoglobin A (deoxy-Hb A), and it has been reported that the functional properties of Hb A in single crystals are different from those in solution. Carbonmonoxyhemoglobin A (HbCO A) can also be crystallized in several structures. Our recent RDC studies of HbCO A in the absence and presence of IHP have shown that the solution structure of this Hb molecule is distinctly different from its classical crystal structures (R and R2). To have a better understanding of the structure-function relationship of Hb A under physiological conditions, we need to evaluate its structures in both ligated and unligated states in solution. Here, the intrinsic paramagnetic property of deoxy-Hb A has been exploited for the measurement of RDCs using the magnetic-field dependence of the apparent one-bond 1H-15N J couplings. Our RDC analysis suggests that the quaternary and tertiary structures of deoxy-Hb A in solution differ from its recently determined high-resolution crystal structures. Upon binding of IHP, structural changes in deoxy-Hb A are also observed, and these changes are largely within the alpha1beta1 (or alpha2beta2) dimer itself. These new structural findings allow us to gain a deeper insight into the structure-function relationship of this interesting allosteric protein.

In recent years paramagnetic NMR derived structural constraints have become increasingly popular for the study of biomolecules. Some of these are based on the distance and angular dependences of pseudo contact shifts (PCSs). When modulated by internal motions PCSs also become sensitive reporters on molecular dynamics. We present here an investigation of the domain-domain motion in a two domain protein (PA0128) through time-modulation of PCSs. PA0128 is a protein of unknown function from Pseudomonas aeruginosa (PA) and contains a Zn(2+) binding site in the N-terminal domain. When substituted with Co(2+) in the binding site, several resonances from the C-terminal domain showed severe line broadening along the (15)N dimension. Relaxation compensated CPMG experiments revealed that the dramatic increase in the (15)N linewidth came from contributions of chemical exchange. Since several sites with perturbed relaxation are localized to a single beta-strand region, and since extracted timescales of motion for the perturbed sites are identical, and since the magnitude of the chemical exchange contributions is consistent with PCSs, the observed rate enhancements are interpreted as the result of concerted domain motion on the timescale of a few milliseconds. Given the predictability of PCS differences and the easy interpretation of the experimental results, we suggest that these effects might be useful in the study of molecular processes occurring on the millisecond to microsecond timescale.

For larger proteins, and proteins not amenable to expression in bacterial hosts, it is difficult to deduce structures using NMR methods based on uniform (13)C, (15)N isotopic labeling and observation of just nuclear Overhauser effects (NOEs). In these cases, sparse labeling with selected (15)N enriched amino acids and extraction of a wider variety of backbone-centered structural constraints is providing an alternate approach. A limitation, however, is the absence of resonance assignment strategies that work without uniform (15)N, (13)C labeling or preparation of numerous samples labeled with pairs of isotopically labeled amino acids. In this paper an approach applicable to a single sample prepared with sparse (15)N labeling in selected amino acids is presented. It relies on correlation of amide proton exchange rates, measured from data on the intact protein and on digested and sequenced peptides. Application is illustrated using the carbohydrate binding protein, Galectin-3. Limitations and future applications are discussed.

The study of bound-state conformations of ligands interacting with proteins is important to the understanding of protein function and the design of drugs that alter function. Traditionally, transferred nuclear Overhauser effects (trNOEs), measured from NMR spectra of ligands in rapid exchange between bound and free states, have been used in these studies, owing to the inherent heavy weighting of bound-state data in the averaged ligand signals. In principle, residual dipolar couplings (RDCs) provide a useful complement to NOE data in that they provide orientational constraints as opposed to distance constraints, but use in ligand-binding applications has been limited due to the absence of heavy weighting of bound-state data. A widely applicable approach to increasing the weighting of bound-state data in averaged RDCs measured on ligands is presented. The approach rests on association of a His-tagged protein with a nickel-chelate-carrying lipid inserted into the lipid bilayer-like alignment media used in the acquisition of RDCs. The approach is validated through the observation of bound-state RDCs for the disaccharide, lactose, bound to the carbohydrate recognition domain of the mammalian lectin, galectin-3.

Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a (15)N phenylalanine-labeled version of the structurally uncharacterized, alpha-2,6-linked sialyltransferase, ST6Gal I.