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A microscale protein NMR sample screening pipeline.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30-200 mug in 8-35 mul volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process step...
Source: Journal of Bimolecular NMR - November 14, 2009 Category: Biomedical Science Authors: Rossi P, Swapna GV, Huang YJ, Aramini JM, Anklin C, Conover K, Hamilton K, Xiao R, Acton TB, Ertekin A, Everett JK, Montelione GT Tags: J Biomol NMR Source Type: journals

Side chain: backbone projections in aromatic and ASX residues from NMR cross-correlated relaxation.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The measurements of cross-correlated relaxation rates between H(N)-N and C(beta)-C(gamma) intraresidual and sequential dipolar interactions is demonstrated in ASN, ASP and aromatic residues. The experiment can be used for deuterated samples and no additional knowledge such as Karplus parametrizations is required for the analysis. The data constitutes a new type of information since no other method relates the C(beta)-C(gamma) bond to H(N)-N. Using this method the dominant populations of rotamer states of chi1 can be readily cross checked provided that phi or psi are known. In addition, dynamics on all timescales can be...
Source: Journal of Bimolecular NMR - November 11, 2009 Category: Biomedical Science Authors: Vögeli B, Riek R Tags: J Biomol NMR Source Type: journals

Comprehensive determination of (3)J (HNHalpha) for unfolded proteins using (13)C'-resolved spin-echo difference spectroscopy.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
An experiment is presented to determine (3)J(HNHalpha) coupling constants, with significant advantages for applications to unfolded proteins. The determination of coupling constants for the peptide chain using 1D (1)H, or 2D and 3D (1)H-(15)N correlation spectroscopy is often hampered by extensive resonance overlap when dealing with flexible, disordered proteins. In the experiment detailed here, the overlap problem is largely circumvented by recording (1)H-(13)C' correlation spectra, which demonstrate superior resolution for unfolded proteins. J-coupling constants are extracted from the peak intensities in a pair of 2D...
Source: Journal of Bimolecular NMR - November 7, 2009 Category: Biomedical Science Authors: Otten R, Wood K, Mulder FA Tags: J Biomol NMR Source Type: journals

TROSY-selected ZZ-exchange experiment for characterizing slow chemical exchange in large proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A TROSY-selected ZZ-exchange experiment is described for measuring slow chemical exchange rates by monitoring the TROSY component of (15)N longitudinal magnetization. Application of the proposed pulse sequence to the cadherin 8 N-terminal extracelluar domain demonstrates that enhanced sensitivity is obtained, compared to a previously described TROSY-detected ZZ-exchange sequence (Sahu et al. J Am Chem Soc 129: 13232-13237, 2007), by preserving the TROSY effect during the mixing period as well as the frequency encoding periods. PMID: 19890725 [PubMed - as supplied by publisher] (Source: Journal of Bimolecular NMR)
Source: Journal of Bimolecular NMR - November 5, 2009 Category: Biomedical Science Authors: Li Y, Palmer AG Tags: J Biomol NMR Source Type: journals

4D prediction of protein (1)H chemical shifts.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A 4D approach for protein (1)H chemical shift prediction was explored. The 4th dimension is the molecular flexibility, mapped using molecular dynamics simulations. The chemical shifts were predicted with a principal component model based on atom coordinates from a database of 40 protein structures. When compared to the corresponding non-dynamic (3D) model, the 4th dimension improved prediction by 6-7%. The prediction method achieved RMS errors of 0.29 and 0.50 ppm for Halpha and HN shifts, respectively. However, for individual proteins the RMS errors were 0.17-0.34 and 0.34-0.65 ppm for the Halpha and HN shifts, respec...
Source: Journal of Bimolecular NMR - October 30, 2009 Category: Biomedical Science Authors: Lehtivarjo J, Hassinen T, Korhonen SP, Peräkylä M, Laatikainen R Tags: J Biomol NMR Source Type: journals

Aliasing in reduced dimensionality NMR spectra: (3,2)D HNHA and (4,2)D HN(COCA)NH experiments as examples.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Reduced dimensionality NMR spectra usually require very large spectral widths in the shared dimension. In this paper we show that aliasing can be introduced in reduced dimensionality NMR spectra either to decrease the acquisition time or increase the resolution of the experiments without losing information. The gains of introducing aliasing are illustrated with two examples, the (3,2)D HNHA and the (4,2)D HN(COCA)NH experiments. In both cases a reduction of the spectral width of more than 50% was possible. PMID: 19851713 [PubMed - as supplied by publisher] (Source: Journal of Bimolecular NMR)
Source: Journal of Bimolecular NMR - October 23, 2009 Category: Biomedical Science Authors: Pantoja-Uceda D, Santoro J Tags: J Biomol NMR Source Type: journals

Simple tests for the validation of multiple field spin relaxation data.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
(15)N spin relaxation data is widely used to extract detailed dynamic information regarding bond vectors such as the amide N-H bond of the protein backbone. Analysis is typically carried using the Lipari-Szabo model-free approach. Even though the original model-free equation can be determined from single field R (1), R (2) and NOE, over-determination of more complex motional models is dependent on the recording of multiple field datasets. This is especially important for the characterization of conformational exchange which affects R (2) in a field dependent manner. However, severe artifacts can be introduced if incons...
Source: Journal of Bimolecular NMR - October 19, 2009 Category: Biomedical Science Authors: Morin S, M Gagné S Tags: J Biomol NMR Source Type: journals

Protein structure calculation with data imputation: the use of substitute restraints.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The amount of experimental restraints e.g., NOEs is often too small for calculating high quality three-dimensional structures by restrained molecular dynamics. Considering this as a typical missing value problem we propose here a model based data imputation technique that should lead to an improved estimation of the correct structure. The novel automated method implemented in AUREMOL makes a more efficient use of the experimental information to obtain NMR structures with higher accuracy. It creates a large set of substitute restraints that are used either alone or together with the experimental restraints. The new appr...
Source: Journal of Bimolecular NMR - October 16, 2009 Category: Biomedical Science Authors: Cano C, Brunner K, Baskaran K, Elsner R, Munte CE, Kalbitzer HR Tags: J Biomol NMR Source Type: journals

Analysis of the amide (15)N chemical shift tensor of the C(alpha) tetrasubstituted constituent of membrane-active peptaibols, the alpha-aminoisobutyric acid residue, compared to those of di- and tri-substituted proteinogenic amino acid residues.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The ...
Source: Journal of Bimolecular NMR - October 10, 2009 Category: Biomedical Science Authors: Salnikov E, Bertani P, Raap J, Bechinger B Tags: J Biomol NMR Source Type: journals

The NMR restraints grid at BMRB for 5,266 protein and nucleic acid PDB entries.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Several pilot experiments have indicated that improvements in older NMR structures can be expected by applying modern software and new protocols (Nabuurs et al. in Proteins 55:483-186, 2004; Nederveen et al. in Proteins 59:662-672, 2005; Saccenti and Rosato in J Biomol NMR 40:251-261, 2008). A recent large scale X-ray study also has shown that modern software can significantly improve the quality of X-ray structures that were deposited more than a few years ago (Joosten et al. in J. Appl Crystallogr 42:376-384, 2009; Sanderson in Nature 459:1038-1039, 2009). Recalculation of three-dimensional coordinates requires that ...
Source: Journal of Bimolecular NMR - October 6, 2009 Category: Biomedical Science Authors: Doreleijers JF, Vranken WF, Schulte C, Lin J, Wedell JR, Penkett CJ, Vuister GW, Vriend G, Markley JL, Ulrich EL Tags: J Biomol NMR Source Type: journals

Isotope labeling strategies for NMR studies of RNA.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The known biological functions of RNA have expanded in recent years and now include gene regulation, maintenance of sub-cellular structure, and catalysis, in addition to propagation of genetic information. As for proteins, RNA function is tightly correlated with structure. Unlike proteins, structural information for larger, biologically functional RNAs is relatively limited. NMR signal degeneracy, relaxation problems, and a paucity of long-range (1)H-(1)H dipolar contacts have limited the utility of traditional NMR approaches. Selective isotope labeling, including nucleotide-specific and segmental labeling strategies, ...
Source: Journal of Bimolecular NMR - September 29, 2009 Category: Biomedical Science Authors: Lu K, Miyazaki Y, Summers MF Tags: J Biomol NMR Source Type: journals

Production of isotopically labeled heterologous proteins in non-E. coli prokaryotic and eukaryotic cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The preparation of stable isotope-labeled proteins is necessary for the application of a wide variety of NMR methods, to study the structures and dynamics of proteins and protein complexes. The E. coli expression system is generally used for the production of isotope-labeled proteins, because of the advantages of ease of handling, rapid growth, high-level protein production, and low cost for isotope-labeling. However, many eukaryotic proteins are not functionally expressed in E. coli, due to problems related to disulfide bond formation, post-translational modifications, and folding. In such cases, other expression syst...
Source: Journal of Bimolecular NMR - September 28, 2009 Category: Biomedical Science Authors: Takahashi H, Shimada I Tags: J Biomol NMR Source Type: journals

Methyl groups as probes of supra-molecular structure, dynamics and function.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The development of new protein labeling strategies, along with optimized experiments that exploit the label, have significantly impacted on the types of biochemical problems that can now be addressed by solution NMR spectroscopy. Here we describe how methyl labeling of key residues in a highly deuterated protein background has facilitated studies of the structure, dynamics and interactions of supra-molecular particles. The methyl-labeling approach is briefly reviewed, followed by a summary of applications to three different molecular machines so as to illustrate the types of questions that can now be addressed. Areas w...
Source: Journal of Bimolecular NMR - September 26, 2009 Category: Biomedical Science Authors: Ruschak AM, Kay LE Tags: J Biomol NMR Source Type: journals

Characterization of different water pools in solid-state NMR protein samples.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We observed and characterized two distinct signals originating from different pools of water protons in solid-state NMR protein samples, namely from crystal water which exchanges polarization with the protein (on the NMR timescale) and is located in the protein-rich fraction at the periphery of the magic-angle spinning (MAS) sample container, and supernatant water located close to the axis of the sample container. The polarization transfer between the water and the protein can be probed by two-dimensional exchange spectroscopy, and we show that the supernatant water does not interact with protein on the timescale of th...
Source: Journal of Bimolecular NMR - September 24, 2009 Category: Biomedical Science Authors: Böckmann A, Gardiennet C, Verel R, Hunkeler A, Loquet A, Pintacuda G, Emsley L, Meier BH, Lesage A Tags: J Biomol NMR Source Type: journals

An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in (15)N, (13)C labeled proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
An improved pulse sequence, intraresidual i(HCA)CO(CA)NH, is described for establishing solely (13)C'(i), (15)N(i), (1)H(N)(i) connectivities in uniformly (15)N/(13)C-labeled proteins. In comparison to the "out-and-back" style intra-HN(CA)CO experiment, the new pulse sequence offers at least two-fold higher experimental resolution in the (13)C' dimension and on average 1.6 times higher sensitivity especially for residues in alpha-helices. Performance of the new experiment was tested on a small globular protein ubiquitin and an intrinsically unfolded 110-residue cancer/testis antigen CT16/PAGE5. Use of intraresidual i(H...
Source: Journal of Bimolecular NMR - September 18, 2009 Category: Biomedical Science Authors: Mäntylahti S, Tossavainen H, Hellman M, Permi P Tags: J Biomol NMR Source Type: journals

Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Although the rapid progress of NMR technology has significantly expanded the range of NMR-trackable systems, preparation of NMR-suitable samples that are highly soluble and stable remains a bottleneck for studies of many biological systems. The application of solubility-enhancement tags (SETs) has been highly effective in overcoming solubility and sample stability issues and has enabled structural studies of important biological systems previously deemed unapproachable by solution NMR techniques. In this review, we provide a brief survey of the development and successful applications of the SET strategy in biomolecular...
Source: Journal of Bimolecular NMR - September 2, 2009 Category: Biomedical Science Authors: Zhou P, Wagner G Tags: J Biomol NMR Source Type: journals

Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in (15)N- (13)C-ILV methyl labeled proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present a time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record (13)C- and (15)N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples. This experiment is designed to delineate the two spectra which would otherwise overlap with one another when acquired together. These spectra display nOe correlations in the detected proton dimension, i.e. with maximum resolution. This is in contrast to NOESY-HSQC types of experiments that provide cross-peaks in the indirect dimension with low resolution due to limits in experimental time. The technique is particularly advantageous at high field whe...
Source: Journal of Bimolecular NMR - September 1, 2009 Category: Biomedical Science Authors: Frueh DP, Leed A, Arthanari H, Koglin A, Walsh CT, Wagner G Tags: J Biomol NMR Source Type: journals

The NMR structure of the p62 PB1 domain, a key protein in autophagy and NF-kappaB signaling pathway.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
PMID: 19728111 [PubMed - as supplied by publisher] (Source: Journal of Bimolecular NMR)
Source: Journal of Bimolecular NMR - August 28, 2009 Category: Biomedical Science Authors: Saio T, Yokochi M, Inagaki F Tags: J Biomol NMR Source Type: journals

High-resolution protein structure determination starting with a global fold calculated from exact solutions to the RDC equations.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present a novel structure determination approach that exploits the global orientational restraints from RDCs to resolve ambiguous NOE assignments. Unlike traditional approaches that bootstrap the initial fold from ambiguous NOE assignments, we start by using RDCs to compute accurate secondary structure element (SSE) backbones at the beginning of structure calculation. Our structure determination package, called RDC-PANDA: (RDC-based SSE PAcking with NOEs for Structure Determination and NOE Assignment), consists of three modules: (1) RDC-EXACT: ; (2) PACKER: ; and (3) HANA: (HAusdorff-based NOE Assignment). RDC-EXACT: co...
Source: Journal of Bimolecular NMR - August 26, 2009 Category: Biomedical Science Authors: Zeng J, Boyles J, Tripathy C, Wang L, Yan A, Zhou P, Donald BR Tags: J Biomol NMR Source Type: journals

FM reconstruction of non-uniformly sampled protein NMR data at higher dimensions and optimization by distillation.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Non-uniform sampling (NUS) enables recording of multidimensional NMR data at resolutions matching the resolving power of modern instruments without using excessive measuring time. However, in order to obtain satisfying results, efficient reconstruction methods are needed. Here we describe an optimized version of the Forward Maximum entropy (FM) reconstruction method, which can reconstruct up to three indirect dimensions. For complex datasets, such as NOESY spectra, the performance of the procedure is enhanced by a distillation procedure that reduces artifacts stemming from intense peaks. PMID: 19705283 [PubMed - as...
Source: Journal of Bimolecular NMR - August 24, 2009 Category: Biomedical Science Authors: Hyberts SG, Frueh DP, Arthanari H, Wagner G Tags: J Biomol NMR Source Type: journals

Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D(2)O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both (1)H and (15)N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for (1)H-(15)N correlations in dipolar coupling based experiments for H(2)O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based (1)H-(15)N correlation experiments yield a nea...
Source: Journal of Bimolecular NMR - August 21, 2009 Category: Biomedical Science Authors: Akbey U, Lange S, Trent Franks W, Linser R, Rehbein K, Diehl A, van Rossum BJ, Reif B, Oschkinat H Tags: J Biomol NMR Source Type: journals

Simultaneous convection compensation and solvent suppression in biomolecular NMR diffusion experiments.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Thermal convection and high intensity solvent resonances can significantly hamper diffusion estimates in pulsed gradient spin-echo nuclear magnetic resonance diffusion experiments on biomolecule samples. To overcome these two problems, a new double functional NMR diffusion sequence, double echo PGSTE-WATERGATE, is presented. The new sequence provides excellent convection compensation and solvent suppression (with a suppression factor in excess of at least 10(5) in a single scan) in biomolecular NMR diffusion experiments. Due to its stimulated echo nature, the new sequence is much less susceptible to spin-spin relaxatio...
Source: Journal of Bimolecular NMR - August 20, 2009 Category: Biomedical Science Authors: Zheng G, Price WS Tags: J Biomol NMR Source Type: journals

Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In the last 15 years substantial advances have been made to place isotope labels in native and glycosylated proteins for NMR studies and structure determination. Key developments include segmental isotope labeling using Native Chemical Ligation, Expressed Protein Ligation and Protein Trans-Splicing. These advances are pushing the size limit of NMR spectroscopy further making larger proteins accessible for this technique. It is just emerging that segmental isotope labeling can be used to define inter-domain interactions in NMR structure determination. Labeling of post-translational modified proteins like glycoproteins r...
Source: Journal of Bimolecular NMR - August 18, 2009 Category: Biomedical Science Authors: Skrisovska L, Schubert M, Allain FH Tags: J Biomol NMR Source Type: journals

Solution structure of the human Tax-interacting protein-1.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
PMID: 19685007 [PubMed - as supplied by publisher] (Source: Journal of Bimolecular NMR)
Source: Journal of Bimolecular NMR - August 14, 2009 Category: Biomedical Science Authors: Durney MA, Birrane G, Anklin C, Soni A, Ladias JA Tags: J Biomol NMR Source Type: journals

Cell-free expression and stable isotope labelling strategies for membrane proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems provides a very interesting alternative to cell-based systems, since it circumvents many problems such as toxicity or necessit...
Source: Journal of Bimolecular NMR - August 12, 2009 Category: Biomedical Science Authors: Sobhanifar S, Reckel S, Junge F, Schwarz D, Kai L, Karbyshev M, Löhr F, Bernhard F, Dötsch V Tags: J Biomol NMR Source Type: journals

Site-specific labeling of proteins with NMR-active unnatural amino acids.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural...
Source: Journal of Bimolecular NMR - August 8, 2009 Category: Biomedical Science Authors: Jones DH, Cellitti SE, Hao X, Zhang Q, Jahnz M, Summerer D, Schultz PG, Uno T, Geierstanger BH Tags: J Biomol NMR Source Type: journals

A topical issue: NMR investigations of molecular dynamics.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
PMID: 19669621 [PubMed - as supplied by publisher] (Source: Journal of Bimolecular NMR)
Source: Journal of Bimolecular NMR - August 8, 2009 Category: Biomedical Science Authors: Palmer AG Tags: J Biomol NMR Source Type: journals

Slight mistuning of a cryogenic probe significantly perturbs the water (1)H precession frequency.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A shift of the water proton precession frequency is described that can introduce errors in chemical shifts derived using the water signal as the chemical shift reference. This shift, f(s), arises as a consequence of radiation damping when the water proton and detector circuit resonance frequencies differ. Herein it is shown that experimental values of f(s), measured as a function of detector circuit tuning offset for 500 and 900 MHz cryogenic probes, are in good agreement with theory. Of importance is the fact that even a small degree of mistuning, which does not significantly impact the performance of a pulse sequence...
Source: Journal of Bimolecular NMR - August 7, 2009 Category: Biomedical Science Authors: Torchia DA Tags: J Biomol NMR Source Type: journals

Hydration dependent dynamics in RNA.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms-ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in (2)H solid...
Source: Journal of Bimolecular NMR - August 7, 2009 Category: Biomedical Science Authors: Olsen GL, Bardaro MF, Echodu DC, Drobny GP, Varani G Tags: J Biomol NMR Source Type: journals

Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL a...
Source: Journal of Bimolecular NMR - August 6, 2009 Category: Biomedical Science Authors: Takeda M, Ono AM, Terauchi T, Kainosho M Tags: J Biomol NMR Source Type: journals

Characterization of slow conformational dynamics in solids: dipolar CODEX.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A solid state NMR experiment is introduced for probing relatively slow conformational exchange, based on dephasing and refocusing dipolar couplings. The method is closely related to the previously described Centerband-Only Detection of Exchange or CODEX experiment. The use of dipolar couplings for this application is advantageous because their values are known a priori from molecular structures, and their orientations and reorientations relate in a simple way to molecular geometry and motion. Furthermore the use of dipolar couplings in conjunction with selective isotopic enrichment schemes is consistent with selection ...
Source: Journal of Bimolecular NMR - August 6, 2009 Category: Biomedical Science Authors: Li W, McDermott AE Tags: J Biomol NMR Source Type: journals

Approaches for the measurement of solvent exposure in proteins by (19)F NMR.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Fluorine NMR is a useful tool to probe protein folding, conformation and local topology owing to the sensitivity of the chemical shift to the local electrostatic environment. As an example we make use of (19)F NMR and 3-fluorotyrosine to evaluate the conformation and topology of the tyrosine residues (Tyr-99 and Tyr-138) within the EF-hand motif of the C-terminal domain of calmodulin (CaM) in both the calcium-loaded and calcium-free states. We critically compare approaches to assess topology and solvent exposure via solvent isotope shifts, (19)F spin-lattice relaxation rates, (1)H-(19)F nuclear Overhauser effects, and ...
Source: Journal of Bimolecular NMR - August 4, 2009 Category: Biomedical Science Authors: Kitevski-Leblanc JL, Evanics F, Prosser RS Tags: J Biomol NMR Source Type: journals

Accessing ns-mus side chain dynamics in ubiquitin with methyl RDCs.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This study presents the first application of the model-free analysis (MFA) (Meiler in J Am Chem Soc 123:6098-6107, 2001; Lakomek in J Biomol NMR 34:101-115, 2006) to methyl group RDCs measured in 13 different alignment media in order to describe their supra-tau (c) dynamics in ubiquitin. Our results indicate that methyl groups vary from rigid to very mobile with good correlation to residue type, distance to backbone and solvent exposure, and that considerable additional dynamics are effective at rates slower than the correlation time tau (c). In fact, the average amplitude of motion expressed in terms of order parameters S...
Source: Journal of Bimolecular NMR - August 3, 2009 Category: Biomedical Science Authors: Farès C, Lakomek NA, Walter KF, Frank BT, Meiler J, Becker S, Griesinger C Tags: J Biomol NMR Source Type: journals

Density functional calculations of chemical shielding of backbone (15)N in helical residues of protein G.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We performed density functional calculations of backbone (15)N chemical shielding tensors in selected helical residues of protein G. Here we describe a computationally efficient methodology to include most of the important effects in the calculation of chemical shieldings of backbone (15)N. We analyzed the role of long-range intra-protein electrostatic interactions by comparing models with different complexity in vacuum and in charge field. Our results show that the dipole moment of the alpha-helix can cause significant deshielding of (15)N; therefore, it needs to be considered when calculating (15)N chemical shielding...
Source: Journal of Bimolecular NMR - July 30, 2009 Category: Biomedical Science Authors: Cai L, Fushman D, Kosov DS Tags: J Biomol NMR Source Type: journals

Partially folded equilibrium intermediate of the villin headpiece HP67 defined by (13)C relaxation dispersion.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Identification and characterization of ensembles of intermediate states remains an important objective in describing protein folding in atomic detail. The 67-residue villin headpiece, HP67, consists of an N-terminal subdomain (residues 10-42) that transiently unfolds at equilibrium under native-like conditions and a highly stable C-terminal subdomain (residues 43-76). The transition between folded and unfolded states of the N-terminal domain has been characterized previously by (15)N NMR relaxation dispersion measurements (Grey et al. in J Mol Biol 355:1078, 2006). In the present work, (13)C spin relaxation was used to...
Source: Journal of Bimolecular NMR - July 30, 2009 Category: Biomedical Science Authors: O'Connell NE, Grey MJ, Tang Y, Kosuri P, Miloushev VZ, Raleigh DP, Palmer AG Tags: J Biomol NMR Source Type: journals

Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The conformational entropy of proteins can make significant contributions to the free energy of ligand binding. NMR spin relaxation enables site-specific investigation of conformational entropy, via order parameters that parameterize local reorientational fluctuations of rank-2 tensors. Here we have probed the conformational entropy of lactose binding to the carbohydrate recognition domain of galectin-3 (Gal3), a protein that plays an important role in cell growth, cell differentiation, cell cycle regulation, and apoptosis, making it a potential target for therapeutic intervention in inflammation and cancer. We used (1...
Source: Journal of Bimolecular NMR - July 29, 2009 Category: Biomedical Science Authors: Diehl C, Genheden S, Modig K, Ryde U, Akke M Tags: J Biomol NMR Source Type: journals

Mesodynamics in the SARS nucleocapsid measured by NMR field cycling.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Protein motions on all timescales faster than molecular tumbling are encoded in the spectral density. The dissection of complex protein dynamics is typically performed using relaxation rates determined at high and ultra-high field. Here we expand this range of the spectral density to low fields through field cycling using the nucleocapsid protein of the SARS coronavirus as a model system. The field-cycling approach enables site-specific measurements of R (1) at low fields with the sensitivity and resolution of a high-field magnet. These data, together with high-field relaxation and heteronuclear NOE, provide evidence f...
Source: Journal of Bimolecular NMR - July 29, 2009 Category: Biomedical Science Authors: Clarkson MW, Lei M, Eisenmesser EZ, Labeikovsky W, Redfield A, Kern D Tags: J Biomol NMR Source Type: journals

Mapping the dynamics of ligand reorganization via (13)CH (3) and (13)CH (2) relaxation dispersion at natural abundance.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Flexible ligands pose challenges to standard structure-activity studies since they frequently reorganize their conformations upon protein binding and catalysis. Here, we demonstrate the utility of side chain (13)C relaxation dispersion measurements to identify and quantify the conformational dynamics that drive this reorganization. The dispersion measurements probe methylene (13)CH(2) and methyl (13)CH(3) groups; the latter are highly prevalent side chain moieties in known drugs. Combining these side chain studies with existing backbone dispersion studies enables a comprehensive investigation of mus-ms conformational d...
Source: Journal of Bimolecular NMR - July 28, 2009 Category: Biomedical Science Authors: Peng JW, Wilson BD, Namanja AT Tags: J Biomol NMR Source Type: journals

Protein proton-proton dynamics from amide proton spin flip rates.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Residue-specific amide proton spin-flip rates K were measured for peptide-free and peptide-bound calmodulin. K approximates the sum of NOE build-up rates between the amide proton and all other protons. This work outlines the theory of multi-proton relaxation, cross relaxation and cross correlation, and how to approximate it with a simple model based on a variable number of equidistant protons. This model is used to extract the sums of K-rates from the experimental data. Error in K is estimated using bootstrap methodology. We define a parameter Q as the ratio of experimental K-rates to theoretical K-rates, where the the...
Source: Journal of Bimolecular NMR - July 27, 2009 Category: Biomedical Science Authors: Weaver DS, Zuiderweg ER Tags: J Biomol NMR Source Type: journals

Preparation, resonance assignment, and preliminary dynamics characterization of residue specific (13)C/ (15)N-labeled elongated DNA for the study of sequence-directed dynamics by NMR.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA is a highly flexible molecule that undergoes functionally important structural transitions in response to external cellular stimuli. Atomic level spin relaxation NMR studies of DNA dynamics have been limited to short duplexes in which sensitivity to biologically relevant fluctuations occurring at nanosecond timescales is often inadequate. Here, we introduce a method for preparing residue-specific (13)C/(15)N-labeled elongated DNA along with a strategy for establishing resonance assignments and apply the approach to probe fast inter-helical bending motions induced by an adenine tract. Preliminary results suggest the...
Source: Journal of Bimolecular NMR - July 27, 2009 Category: Biomedical Science Authors: Nikolova EN, Al-Hashimi HM Tags: J Biomol NMR Source Type: journals

Monitoring conformational dynamics with solid-state R (1rho) experiments.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A new application of solid-state rotating frame (R (1rho)) relaxation experiments to observe conformational dynamics is presented. Studies on a model compound, dimethyl sulfone (DMS), show that R (1rho) relaxation due to reorientation of a chemical shift anisotropy (CSA) tensor undergoing chemical exchange can be used to monitor slow-to-intermediate timescale conformational exchange processes. Control experiments used d ( 6 ) -DMS and alanine to confirm that the technique is monitoring reorientation of the CSA tensor rather than dipolar interactions or methyl group rotation. The application of this method to proteins c...
Source: Journal of Bimolecular NMR - July 27, 2009 Category: Biomedical Science Authors: Quinn CM, McDermott AE Tags: J Biomol NMR Source Type: journals

RNA phosphodiester backbone dynamics of a perdeuterated cUUCGg tetraloop RNA from phosphorus-31 NMR relaxation analysis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We have analyzed the relaxation properties of all (31)P nuclei in an RNA cUUCGg tetraloop model hairpin at proton magnetic field strengths of 300, 600 and 900 MHz in solution. Significant H, P dipolar contributions to R (1) and R (2) relaxation are observed in a protonated RNA sample at 600 MHz. These contributions can be suppressed using a perdeuterated RNA sample. In order to interpret the (31)P relaxation data (R (1), R (2)), we measured the (31)P chemical shift anisotropy (CSA) by solid-state NMR spectroscopy under various salt and hydration conditions. A value of 178.5 ppm for the (31)P CSA in the static state (S ...
Source: Journal of Bimolecular NMR - July 27, 2009 Category: Biomedical Science Authors: Rinnenthal J, Richter C, Nozinovic S, Fürtig B, Lopez JJ, Glaubitz C, Schwalbe H Tags: J Biomol NMR Source Type: journals

AMORE-HX: a multidimensional optimization of radial enhanced NMR-sampled hydrogen exchange.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The Cartesian sampled three-dimensional HNCO experiment is inherently limited in time resolution and sensitivity for the real time measurement of protein hydrogen exchange. This is largely overcome by use of the radial HNCO experiment that employs the use of optimized sampling angles. The significant practical limitation presented by use of three-dimensional data is the large data storage and processing requirements necessary and is largely overcome by taking advantage of the inherent capabilities of the 2D-FT to process selective frequency space without artifact or limitation. Decomposition of angle spectra into posit...
Source: Journal of Bimolecular NMR - July 25, 2009 Category: Biomedical Science Authors: Gledhill JM, Walters BT, Wand AJ Tags: J Biomol NMR Source Type: journals

Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The molecular motions of membrane proteins in liquid-crystalline lipid bilayers lie at the interface between motions in isotropic liquids and in solids. Specifically, membrane proteins can undergo whole-body uniaxial diffusion on the microsecond time scale. In this work, we investigate the (1)H rotating-frame spin-lattice relaxation (T (1rho)) caused by the uniaxial diffusion of the influenza A M2 transmembrane peptide (M2TMP), which forms a tetrameric proton channel in lipid bilayers. This uniaxial diffusion was proved before by (2)H, (15)N and (13)C NMR lineshapes of M2TMP in DLPC bilayers. When bound to an inhibitor...
Source: Journal of Bimolecular NMR - July 24, 2009 Category: Biomedical Science Authors: Cady SD, Hong M Tags: J Biomol NMR Source Type: journals

Quantitative analysis of backbone motion in proteins using MAS solid-state NMR spectroscopy.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present a comprehensive analysis of protein dynamics for a micro-crystallin protein in the solid-state. Experimental data include (15)N T (1) relaxation times measured at two different magnetic fields as well as (1)H-(15)N dipole, (15)N CSA cross correlated relaxation rates which are sensitive to the spectral density function J(0) and are thus a measure of T (2) in the solid-state. In addition, global order parameters are included from a (1)H,(15)N dipolar recoupling experiment. The data are analyzed within the framework of the extended model-free Clore-Lipari-Szabo theory. We find slow motional correlation times in the...
Source: Journal of Bimolecular NMR - July 23, 2009 Category: Biomedical Science Authors: Chevelkov V, Fink U, Reif B Tags: J Biomol NMR Source Type: journals

Structural dynamics of protein backbone phi angles: extended molecular dynamics simulations versus experimental (3) J scalar couplings.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
(3) J scalar couplings report on the conformational averaging of backbone phi angles in peptides and proteins, and therefore represent a potentially powerful tool for studying the details of both structure and dynamics in solution. We have compared an extensive experimental dataset with J-couplings predicted from unrestrained molecular dynamics simulation using enhanced sampling available from accelerated molecular dynamics or using long timescale trajectories (200 ns). The dynamic fluctuations predicted to be present along the backbone, in agreement with residual dipolar coupling analysis, are compatible with the expe...
Source: Journal of Bimolecular NMR - July 23, 2009 Category: Biomedical Science Authors: Markwick PR, Showalter SA, Bouvignies G, Brüschweiler R, Blackledge M Tags: J Biomol NMR Source Type: journals

Chemical exchange effects during refocusing pulses in constant-time CPMG relaxation dispersion experiments.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In the analysis of the constant-time Carr-Purcell-Meiboom-Gill (CT-CPMG) relaxation dispersion experiment, chemical exchange parameters, such as rate of exchange and population of the exchanging species, are typically optimized using equations that predict experimental relaxation rates recorded as a function of effective field strength. In this process, the effect of chemical exchange during the CPMG pulses is typically assumed to be the same as during the free-precession. This approximation may introduce systematic errors into the analysis of data because the number of CPMG pulses is incremented during the constant-ti...
Source: Journal of Bimolecular NMR - July 18, 2009 Category: Biomedical Science Authors: Myint W, Ishima R Tags: J Biomol NMR Source Type: journals

Probing the urea dependence of residual structure in denatured human alpha-lactalbumin.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Backbone (15)N relaxation parameters and (15)N-(1)H(N) residual dipolar couplings (RDCs) have been measured for a variant of human alpha-lactalbumin (alpha-LA) in 4, 6, 8 and 10 M urea. In the alpha-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala alpha-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. (15)N R(2) values in some regions of all-Ala alpha-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental ...
Source: Journal of Bimolecular NMR - July 18, 2009 Category: Biomedical Science Authors: Higman VA, Rösner HI, Ugolini R, Greene LH, Redfield C, Smith LJ Tags: J Biomol NMR Source Type: journals

Assigning large proteins in the solid state: a MAS NMR resonance assignment strategy using selectively and extensively (13)C-labelled proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared...
Source: Journal of Bimolecular NMR - July 16, 2009 Category: Biomedical Science Authors: Higman VA, Flinders J, Hiller M, Jehle S, Markovic S, Fiedler S, van Rossum BJ, Oschkinat H Tags: J Biomol NMR Source Type: journals

Automated NMR structure determination of stereo-array isotope labeled ubiquitin from minimal sets of spectra using the SAIL-FLYA system.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Stereo-array isotope labeling (SAIL) has been combined with the fully automated NMR structure determination algorithm FLYA to determine the three-dimensional structure of the protein ubiquitin from different sets of input NMR spectra. SAIL provides a complete stereo- and regio-specific pattern of stable isotopes that results in sharper resonance lines and reduced signal overlap, without information loss. Here we show that as a result of the superior quality of the SAIL NMR spectra, reliable, fully automated analyses of the NMR spectra and structure calculations are possible using fewer input spectra than with conventio...
Source: Journal of Bimolecular NMR - July 13, 2009 Category: Biomedical Science Authors: Ikeya T, Takeda M, Yoshida H, Terauchi T, Jee JG, Kainosho M, Güntert P Tags: J Biomol NMR Source Type: journals