Biomolecular NMR Research Articles

Spatially encoded strategies in the execution of biomolecular-oriented 3D NMR experiments

Three-dimensional nuclear magnetic resonance (3D NMR) provides one of the foremost analytical tools available for the elucidation of biomolecular structure, function and dynamics. Executing a 3D NMR experiment generally involves scanning a series of time-domain signals S(t3), as a function of two time variables (t1, t2) which need to undergo parametric incrementations throughout independent experiments. Recent years have witnessed extensive efforts towards the acceleration of this kind of experiments. Among the different approaches that have been proposed counts an "ultrafast" scheme, which distinguishes itself from other propositions by enabling--at least in principle--the acquisition of the complete multidimensional NMR data set within a single transient. 2D protein NMR implementations of this single-scan method have been demonstrated, yet its potential for 3D acquisitions has only been exemplified on model organic compounds. This publication discusses a number of strategies that could make these spatial encoding protocols compatible with 3D biomolecular NMR applications. These include a merging of 2D ultrafast NMR principles with temporal 2D encoding schemes, which can yield 3D HNCO spectra from peptides and proteins within approximately 100 s timescales. New processing issues that facilitate the collection of 3D NMR spectra by relying fully on spatial encoding principles are also assessed, and shown capable of delivering HNCO spectra within 1 s timescales. Limitations and prospects of these various schemes are briefly addressed.

Engineered Sarafotoxins as Tissue Inhibitor of Metalloproteinases-like Matrix Metalloproteinase Inhibitors

The sarafotoxins and endothelins are approximately 25-residue peptides that spontaneously fold into a defined tertiary structure with specific pairing of four cysteines into two disulfide bonds. Their structures show an interesting topological similarity to the core of the metalloproteinase interaction sites of the tissue inhibitors of metalloproteinases. Previous work indicates that sarafotoxins and endothelins can be engineered to eliminate or greatly reduce their vasopressive action and that their structural framework can withstand multiple sequence changes. When sarafotoxin 6b, which possesses modest matrix metalloproteinase inhibitory activity, was C-terminally truncated to remove its toxic vasopressive activity, the metalloproteinase inhibitory activity was essentially abolished. However, further changes, based on the sequences of peptides selected from libraries of sarafotoxin variants or suggested by analogy with tissue inhibitors of metalloproteinases, progressively enhanced the matrix metalloproteinase inhibitory activity. Peptide variants with multiple substitutions folded correctly and formed native disulfide bonds. Improvements in matrix metalloproteinase affinity have generated a peptide with micromolar K(i) values for matrix metalloproteinase-1 and -9 that are selective inhibitors of different metalloproteinases. Characterization of its solution structure indicates a close similarity to sarafotoxin but with a more extended C-terminal helix. The effects of N-acetylation and other changes, as well as docking studies, support the hypothesis that the engineered sarafotoxins bind to matrix metalloproteinases in a manner analogous to the tissue inhibitors of metalloproteinases.

Perspectives of biomolecular NMR in drug discovery: the blessing and curse of versatility

The versatility of NMR and its broad applicability to several stages in the drug discovery process is well known and generally considered one of the major strengths of NMR (Pellecchia et al., Nature Rev Drug Discov 1:211-219, 2002; Stockman and Dalvit, Prog Nucl Magn Reson Spectrosc 41:187-231, 2002; Lepre et al., Comb Chem High throughput screen 5:583-590, 2002; Wyss et al., Curr Opin Drug Discov Devel 5:630-647, 2002; Jahnke and Widmer, Cell Mol Life Sci 61:580-599, 2004; Huth et al., Methods Enzymol 394:549-571, 2005b; Klages et al., Mol Biosyst 2:318-332, 2006; Takeuchi and Wagner, Curr Opin Struct Biol 16:109-117, 2006; Zartler and Shapiro, Curr Pharm Des 12:3963-3972, 2006). Indeed, NMR is the only biophysical technique which can detect and quantify molecular interactions, and at the same time provide detailed structural information with atomic level resolution. NMR should therefore be ideally suited and widely requested as a tool for drug discovery research, and numerous examples of drug discovery projects which have substantially benefited from NMR contributions or were even driven by NMR have been described in the literature. However, not all pharmaceutical companies have rigorously implemented NMR as integral tool of their research processes. Some companies invest with limited resources, and others do not use biomolecular NMR at all. This discrepancy in assessing the value of a technology is striking, and calls for clarification--under which circumstances can NMR provide added value to the drug discovery process? What kind of contributions can NMR make, and how is it implemented and integrated for maximum impact? This perspectives article suggests key areas of impact for NMR, and a model of integrating NMR with other technologies to realize synergies and maximize their value for drug discovery.

Spin amplification in solution magnetic resonance using radiation damping

The sensitive detection of dilute solute spins is critical to biomolecular NMR. In this work, a spin amplifier for detecting dilute solute magnetization is developed using the radiation damping interaction in solution magnetic resonance. The evolution of the solvent magnetization, initially placed along the unstable -z direction, is triggered by the radiation damping field generated by the dilute solute magnetization. As long as the radiation damping field generated by the solute is larger than the corresponding thermal noise field generated by the sample coil, the solute magnetization can effectively trigger the evolution of the water magnetization under radiation damping. The coupling between the solute and solvent magnetizations via the radiation damping field can be further improved through a novel bipolar gradient scheme, which allows solute spins with chemical shift differences much greater than the effective radiation damping field strength to affect the solvent magnetizations more efficiently. Experiments performed on an aqueous acetone solution indicate that solute concentrations on the order of 10(-5) that of the solvent concentration can be readily detected using this spin amplifier.

An automated tool for maximum entropy reconstruction of biomolecular NMR spectra

An automated tool for maximum entropy reconstruction of biomolecular NMR spectra

Structural Basis for Spinophilin−Neurabin Receptor Interaction

Neurabin and spinophilin are neuronal scaffolding proteins that play important roles in the regulation of synaptic transmission through their ability to target protein phosphatase 1 (PP1) to dendritic spines where PP1 dephosphorylates and inactivates glutamate receptors. However, thus far, it is still unknown how neurabin and spinophilin themselves are targeted to these membrane receptors. Spinophilin and neurabin contain a single PDZ domain, a common protein-protein interaction recognition motif, which are 86% identical in sequence. We report the structures of both the neurabin and spinophilin PDZ domains determined using biomolecular NMR spectroscopy. These proteins form the canonical PDZ domain fold. However, despite their high degree of sequence identity, there are distinct and significant structural differences between them, especially between the peptide binding pockets. Using two-dimensional 1H-15N HSQC NMR analysis, we demonstrate that C-terminal peptide ligands derived from glutamatergic AMPA and NMDA receptors and cytosolic proteins directly and differentially bind spinophilin and neurabin PDZ domains. This peptide binding data also allowed us to classify the neurabin and spinophilin PDZ domains as the first identified neuronal hybrid class V PDZ domains, which are capable of binding both class I and II peptides. Finally, the ability to bind to glutamate receptor subunits suggests that the PDZ domains of neurabin and spinophilin are important for targeting PP1 to C-terminal phosphorylation sites in AMPA and NMDA receptor subunits.

Accessible NMR experiments studying the hydrodynamics of 15N‐enriched ubiquitin at low fields

We have recently developed and implemented two experiments in biomolecular NMR for an undergraduate-level biophysical chemistry laboratory with commercially available (15) N-enriched human ubiquitin. These experiments take advantage of (15) N direct detection of the NMR signal. The first experiment develops skills in acquiring and interpreting one-dimensional and two-dimensional NMR data of an aqueous protein sample (D. Rovnyak, L. E. Thompson (2005) An accessible two-dimensional solution nuclear magnetic resonance experiment on human ubiquitin, Biochem. Mol. Biol. Educ. 33, 117-122). In the second experiment, discussed here, students examine the overall dynamics of a protein in solution. Students measure spin relaxation times T(1) and T(2) and use these data to estimate a rotational correlation time, τ(c) , and the diameter of ubiquitin using the Stokes-Einstein relation. This is a fast and simple experiment, requiring about 1 h of instrument time, and a valuable opportunity to provide a hands-on experience in studying macromolecular size and dynamics. This experiment is conducted in synchrony with lecture material on hydrodynamics and sedimentation.

Targeted Acquisition for Real-Time NMR Spectroscopy

A target-oriented approach for the acquisition of information in biomolecular NMR spectroscopy is being developed. This approach combines concurrent data accumulation, processing, and monitoring of spectral quality. Real-time estimation of parameters allows acquisition to be stopped when results are complete and have a specified precision. The technique is based on multidimensional decomposition, which can process incomplete data. An incremental nonuniform sampling scheme ensures the optimization of resolution sensitivity. To validate this method, 3D HNCO spectra of three biomolecular systems (8 kDa ubiquitin, 22 kDa barstar-barnase complex, and 82 kDa malate synthase G) are processed incrementally at small acquisition time steps. The range of molecular sizes illustrates applicability in both sample- and sensitivity-limited regimes. In each case, the target was to acquire all backbone resonances in the spectra. For the three systems, the targets are achieved after 4.5 min, 1.6 h, and 22 h of acquisition time, respectively. A number of other targets that can be similarly monitored as a function of time are discussed.

Cogwheel phase cycling in common triple resonance NMR experiments for the liquid phase

We demonstrate that cogwheel phase cycling, which was previously only used in solid state NMR, can be applied to optimize the efficiency of commonly used pulse sequences in multiple resonance liquid-state biomolecular NMR. In favorable cases the required minimum number of scans can be reduced by more than 80% as compared to a corresponding sequence with nested phase cycles. Since cogwheel phase cycling procedures can be designed for a range of scan numbers, and can be combined with pulsed field gradients, the total experiment time can be adjusted closely to the required signal-to-noise ratio with minimal overhead. Examples are shown for 3D-TROSY-HNCO, 3D-TROSY-HNCACO, and 3D-HACACO experiments on diamagnetic and paramagnetic proteins.

Correlated dynamics of consecutive residues reveal transient and cooperative unfolding of secondary structure in proteins

Nuclear spin relaxation is a powerful method for studying molecular dynamics at atomic resolution. Recent methods development in biomolecular NMR spectroscopy has enabled detailed investigations of molecular dynamics that are critical for biological function, with prominent examples addressing allostery, enzyme catalysis, and protein folding. Dynamic processes with similar correlation times are often detected in multiple locations of the molecule, raising the question of whether the underlying motions are correlated (corresponding to concerted fluctuations involving many atoms distributed across extended regions of the molecule) or uncorrelated (corresponding to independent fluctuations involving few atoms in localized regions). Here, we have used (13)C(alpha)(i - 1)/(13)C(alpha)(i) differential multiple-quantum spin relaxation to provide direct evidence for correlated dynamics of consecutive amino acid residues in the protein sequence. By monitoring overlapping pairs of residues (i - 1 and i, i and i + 1, etc.), we identified correlated motions that extend through continuous segments of the sequence. We detected significant correlated conformational transitions in the native state of the E140Q mutant of the calmodulin C-terminal domain. Previous work has shown that this domain exchanges between two major conformational states that resemble the functionally relevant open and closed states of the WT protein, with a mean correlation time of approximately 20 micros. The present results reveal that an entire alpha-helix undergoes partial unraveling in a transient and cooperative manner.

Definition of a New Information-Based Per-Residue Quality Parameter

For biomolecular NMR structures typically only a poor correspondence is observed between statistics derived from the experimental input data and structural quality indicators obtained from the structure ensembles. Here, we investigate the relationship between the amount of available NMR data and structure quality. By generating datasets with a predetermined information content and evaluating the quality of the resulting structure ensembles we show that there is, in contrast to previous findings, a linear relation between the information contained in experimental data and structural quality. From this relation, a new quality parameter is derived that provides direct insight, on a per-residue basis, into the extent to which structural quality is governed by the experimental input data.

Preview: ChemBioChem 9/2005

ChemBioChemVolume 6, Issue 9 p. 1706-1706 PreviewFree Access Preview: ChemBioChem 9/2005 First published: 01 September 2005 https://doi.org/10.1002/cbic.200590031AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume6, Issue9Special Issue: Biomolecular NMR SpectroscopySeptember 5, 2005Pages 1706-1706 RelatedInformation

Bioelectronics—From Theory to Applications. Edited by Itamar Willner and Eugenii Katz.

ChemBioChemVolume 6, Issue 9 p. 1702-1702 Book Review Bioelectronics—From Theory to Applications. Edited by Itamar Willner and Eugenii Katz. Shana Kelley, Shana Kelley Boston College (US)Search for more papers by this author Shana Kelley, Shana Kelley Boston College (US)Search for more papers by this author First published: 01 September 2005 https://doi.org/10.1002/cbic.200500305Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume6, Issue9Special Issue: Biomolecular NMR SpectroscopySeptember 5, 2005Pages 1702-1702 RelatedInformation

Solid-state NMR in drug design and discovery for membrane-embedded targets

Observing drugs and ligands at their site of action in membrane proteins is now possible through the use of a development in biomolecular NMR spectroscopy known as solid-state NMR. Even large, functionally active complexes are being examined using this method, with structural details being resolved at super-high subnanometre resolution. This is supplemented by detailed dynamic and electronic information about the surrounding ligand environment, and gives surprising new insights into the way in which ligands bind, which can aid drug design.

Orientational Properties of Stretched Polystyrene Gels in Organic Solvents and the Suppression of Their Residual 1H NMR Signals

While residual dipolar couplings (RDCs) are an established method in high-resolution biomolecular NMR, their use for structure determination of small molecules in organic solvents is limited by the alignment media available. Only recently stretched polystyrene (PS) gels were introduced for the measurement of RDCs on small compounds that allowed urgently needed free scalability of the induced anisotropy. Here, the properties of such stretched PS gels in different organic solvents as well as for different magnetic field strengths and temperatures are studied and practical NMR-spectroscopic aspects are discussed.

Reverse Micelles Dissolved in Supercritical Xenon: An NMR Spectroscopic Study

Reverse micelles currently gain increasing interest in chemical technology. They also become important in biomolecular NMR due to their ability to host biomolecules such as proteins. In the present paper, a procedure for the preparation of high-pressure NMR samples containing reverse micelles dissolved in supercritical xenon is presented. These reverse micelles are formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT). For the first time, NMR spectroscopy could be applied to reverse micelles in supercritical xenon. The AOT/H(2)O/Xe system was studied as a function of experimental parameters such as xenon pressure, water content, and salt concentration. Optimum conditions for reverse micelle formation in supercritical xenon could be determined. It is, furthermore, demonstrated that biomolecules such as amino acids and proteins can be incorporated into the reverse micelles dissolved in supercritical xenon.

Human Atherosclerotic Plaque Contains Viable Invasive Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

To the Editor: Because epidemiological evidence supports an association between cardiovascular and periodontal disease, we assessed whether periodontal pathogens were present in atherosclerotic lesions. To detect invasive bacteria, the natural tropism of the bacteria toward human tissues was exploited. Further, bacterial presence was demonstrated using quantitative polymerase chain reaction (Q-PCR). This confirms the presence of periodontal pathogens in atherosclerotic lesions, whereby the bacteria could contribute to the vascular pathology either directly through their cytotoxicity or indirectly by inducing or exacerbating inflammation. Cardiovascular disease (CVD) is the leading cause of death in the in the United States.1 According to the American Heart Association’s statistics from 2003, there were no previous symptoms in 50% of men and 63% of women who died suddenly from CHD. In a 10-year follow-up study, ≈25% of coronary deaths in males and 15% in females occurred in persons in the lowest two quintiles of the multivariate Framingham Heart Study risk scores.2 This and other data have led to an emerging paradigm shift from coronary heart disease having a purely hereditary/nutritional causation to possibly having an infectious component.3 Many epidemiological studies strongly suggest that periodontitis may be a risk factor for coronary heart disease (CHD).4 Serologically, edentulousness and serum IgG-antibodies to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in 1163 men were recently shown to be associated with CHD.5 In a larger prospective study of 6950 subjects, the same authors provide serological evidence that an infection caused by major periodontal pathogens is associated with future stroke.6 Previous studies have identified 16S rRNA of oral microbial pathogens, including P gingivalis and A actinomycetemcomitans , …

Introduction

The 45th annual Sanibel Symposium, organized by the faculty, students, and staff of the Quantum Theory Project of the University of Florida, was held on March 5–11, 2005. More than 300 scientists gathered at a new site, the King and Prince Beach and Golf Resort on St. Simons Island, Georgia. This venue provided excellent conference facilities, and its location on the beach brought back some of the ambiance of the original conference site on Sanibel Island, Florida. The 2005 symposium followed the established format with plenary and poster sessions. A compact six-day integrated program of quantum biology, quantum chemistry, and condensed matter physics provided for intense and lively cross-disciplinary interactions. The meeting honored Enrico Clementi and his scientific contributions. Three sessions on the final day of the symposium were specially dedicated to this celebration and featured as speakers past and present collaborators of Dr. Clementi. The topics of the sessions covered by these proceedings include Protein Design, Large-Scale Simulations of Condensed Phase Systems, Biomolecular NMR, Metals in Biology, DFT and Magnetic Fields, Dynamical Mean Field Theory, High-Level Electron Correlation Theory, Semiempirical Electronic Structure, Coupled-Cluster Theory, Explicitly Correlated and Multi-Resolution Quantum Chemical Calculations, Relativistic Quantum Mechanics, Multi-Reference CC, and MBPT Theory. Participating students were introduced to the conference subjects by well-attended tutorials offered in the evening of the opening Saturday by Professors A. Roitberg and Nigel Richards, and on Monday evening by Professors S. B. Trickey, and So Hirata. The articles in these proceedings have been subjected to the ordinary refereeing procedures of The International Journal of Quantum Chemistry. The organizers acknowledge the following sponsors for their support of the 2005 Sanibel Symposium: US Army (ARO), grant W911NF-05-1-0080; IBM Corporation; and the University of Florida. Very special thanks go to the staff of the Quantum Theory Project of the University of Florida for handling the numerous administrative, clerical, and practical details. The organizers are proud to recognize the contributions of Mrs. Judy Parker, Ms. Coralu Clements, Mr. Jim Folks, and Dr. Ashley Alford. All the graduate students of the Quantum Theory Project, who served as “gofers” are gratefully recognized for their contributions to the 2005 Sanibel Symposium.

Stretched Poly(dimethylsiloxane) Gels as NMR Alignment Media for Apolar and Weakly Polar Organic Solvents: An Ideal Tool for Measuring RDCs at Low Molecular Concentrations

The measurement of residual dipolar couplings (RDCs), meanwhile a standard method for obtaining structural information in biomolecular NMR, requires partial alignment of the sample. Special demands on alignment media so far limit the applicability of this approach to small molecules in organic solvents. Major limitations are the free scalability of alignment and the suppression of residual signals of the alignment medium to allow effective measurement of low-concentration samples. Here, we present stretched poly(dimethylsiloxane) (PDMS) cross-linked by beta-rays as an alignment medium with no visible impurities in 1H NMR spectra but a single signal at approximately 0.1 ppm that can easily be removed by slightly modified water suppression methods. Besides the free scalability, its applicability to the measurement of RDCs in small molecules at low concentration is demonstrated on a approximately 12 mM sample of spiroindene. The induced alignment tensor in this case can be predicted reasonably well by a simplified model on the basis of steric interactions only.

Indirect covariance NMR spectroscopy.

A novel NMR scheme is presented that establishes homonuclear spin correlations without requiring direct detection of the spin species. This covariance NMR method is experimentally demonstrated for a mixture of amino acids and for the uniformly 13C-labeled cyclic decapeptide antamanide using a 13C-edited TOCSY experiment. The method opens up new avenues for the experimental analysis of molecules containing insensitive spins encountered in biomolecular NMR and analytical chemistry including metabolomics.