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Lewis E. Kay - One of the best experts on this subject based on the ideXlab platform.
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The evolution of solution state NMR pulse sequences through the ‘eyes’ of Triple-Resonance spectroscopy
Journal of magnetic resonance (San Diego Calif. : 1997), 2019Co-Authors: Lewis E. KayAbstract:Abstract Careful pulse sequence design and optimization is critical to the success of a given NMR experiment. Over the past several decades the level of sophistication of NMR pulse sequences has increased tremendously, leading to large spectral sensitivity and resolution improvements, to data sets with far fewer artifacts, and to much more rapid acquisition times, opening up a wide range of applications. Here I briefly highlight how pulse sequence ‘engineering’ has evolved, focusing on liquid state NMR, and, in particular, on the HNCA-class of Triple-Resonance experiment. In many respects, the evolution of Triple-Resonance NMR mirrors the evolution of solution state NMR experiments in general, with ‘tricks’ that first appeared in Triple-Resonance pulse sequences or that were motivated by them now incorporated into a broad range of experiments.
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Triple Resonance-based ^13C^α and ^13C^β CEST experiments for studies of ms timescale dynamics in proteins
Journal of Biomolecular NMR, 2014Co-Authors: Dong Long, Ashok Sekhar, Lewis E. KayAbstract:A pair of Triple Resonance based CEST pulse schemes are presented for measuring ^13C^α and ^13C^β chemical shifts of sparsely populated and transiently formed conformers that are invisible to traditional NMR experiments. CEST profiles containing dips at Resonance positions of ^13C^α or ^13C^β spins of major (ground) and minor (excited) conformers are obtained in a pseudo 3rd dimension that is generated by quantifying modulations of cross peaks in ^15N, ^1H^N correlation spectra. An application to the folding reaction of a G48A mutant of the Fyn SH3 domain is presented, illustrating and validating the methodology.
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trosy Triple Resonance four dimensional nmr spectroscopy of a 46 ns tumbling protein
Journal of the American Chemical Society, 1999Co-Authors: Daiwen Yang, Lewis E. KayAbstract:Two four-dimensional TROSY Triple Resonance based experiments are presented for backbone assignment of high molecular weight proteins and protein complexes. The experiments, 4D-HNCACO and 4D-HNCOCA, establish correlations of the form (13Cα(i,i-1),13C‘(i,i-1),15N(i),HN(i)) and (13Cα(i-1),13C‘(i-1),15N(i),HN(i)), respectively. Both sequences use an implementation of TROSY that offers improved sensitivity relative to previous sequences, critical for application to systems with correlation times on the order of 40−50 ns. The utility of the experiments is demonstrated by an application to a 46 ns tumbling complex of the 370 residue maltose binding protein and β-cyclodextrin. Approximately 95% of the expected intra- and interresidue correlations are observed in the HNCACO and HNCOCA, respectively, with average signal-to-noise values of approximately 35/1. The methodology promises to be particularly powerful for applications to high molecular weight complexes comprised of a labeled fragment and unlabeled compone...
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improved 1hn detected Triple Resonance trosy based experiments
Journal of Biomolecular NMR, 1999Co-Authors: Daiwen Yang, Lewis E. KayAbstract:A pulse scheme resulting in improved sensitivity in TROSY-based 1HN-detected Triple Resonance experiments is presented. The approach minimizes relaxation losses which occur during the transfer of transverse magnetization from 15N to 1HN immediately prior to detection. The utility of the method is demonstrated on a complex of methyl protonated, highly deuterated maltose binding protein (MBP, 370 residues) and β- cyclodextrin. Sensitivity gains relative to previous TROSY schemes of approximately 10 and 20% are noted in HNCO spectra of MBP recorded at 25 and 5 °C, respectively, corresponding to molecular correlation times of 23 and 46 ns.
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a multidimensional nmr experiment for measurement of the protein dihedral angle psi based on cross correlated relaxation between 1halpha 13calpha d ipolar and 13c carbonyl chemical shift anisotropy mechanisms
Journal of the American Chemical Society, 1997Co-Authors: Daiwen Yang, Robert Konrat, Lewis E. KayAbstract:A high-resolution Triple-Resonance NMR method is presented for the measurement of the protein backbone dihedral angle ψ based on cross-correlated relaxation between 1Hα−13Cα dipolar and 13C‘ (carbo...
Ad Bax - One of the best experts on this subject based on the ideXlab platform.
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Triple Resonance three dimensional protein nmr before it became a black box
Journal of Magnetic Resonance, 2011Co-Authors: Ad BaxAbstract:Three-dimensional Triple Resonance experiments have become an integral part of virtually every solution NMR study of proteins. The approach relies on uniform isotopic enrichment of proteins with (13)C and (15)N, and establishes the scalar connectivity pathway between nuclei through the large (1)J(NH), (1)J(CH)(, 1)J(CC), and (1)J(CN) couplings. The magnetization transfer process takes place through multiple, efficient one-bond magnetization transfer steps, rather than a single step through the smaller and variable (3)J(HH) couplings. The relatively large size and good uniformity of the one-bond couplings allowed the design of efficient magnetization transfer schemes that are effectively uniform across a given protein, nearly independent of conformation. Although conceptually straightforward, practical implementation of three-dimensional Triple Resonance experiments on proteins originally posed serious challenges. This account provides a personal perspective on some of the historical background to this work, the problems encountered as well as their solutions, and their evolution into today's standard arsenal of experiments.
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carbon 13 line narrowing by deuterium decoupling in deuterium carbon 13 nitrogen 15 enriched proteins application to Triple Resonance 4d j connectivity of sequential amides
Journal of the American Chemical Society, 1993Co-Authors: Stephan Grzesiek, Jacob Anglister, Hao Ren, Ad BaxAbstract:Short transverse I3C relaxation times, T2, constitute the principal barrier for the application of heteronuclear Jcorrelation N M R techniques to larger proteins uniformly enriched with I3C and 15N.1-6 The 13C T2 is dominated by the strong dipolar interaction with its attached protons.' As themagnetogyricratio of 2H is -6.5 times lower than that of IH, the heteronuclear dipolar interaction is greatly reduced by deuteration. Because of the large 2H quadrupolar interaction (170 kHz), the 2H spin lattice relaxation time, TI, in proteins is in the millisecond range at a magnetic field strength of 14 T. Therefore, the 2H-13C J coupling (-22 Hz) does not result in the Triplet shape, expected for a 13C nucleus coupled to a spin-1 nucleus, but gives rise to a collapsed singlet Resonance that is broadened by scalar relaxation of the second k i r ~ d . ~ , ~ High-power (-2.5 W) 2H decoupling with an R F field strength much stronger than the inverse 2H TI effectively removes this broadening and results in a 13C line width that is much narrower than for the protonated I3C. One of the Triple Resonance Jcorrelation experiments affected most by the 13C line width is the H(CA)NH experimenti0%] I which relies on magnetization transfer from Ca to the backbone I5N nucleus via the relatively small I J N c ~ (1 1 Hz) and 2 J ~ ~ a (5-8 Hz) couplings. Although experiments have been proposed to alleviate this difficult J correlation step,l3~l4 the sequential assignment procedure which is based on J correlation between the intraresidue IH/l5N and IHa/l3Ca Resonances and between the 1Hm/l3Ca of residue i and IH/lSN of residue i + 1 is complicated by the high degree of overlap among IHa/l3Ca correlations. Here we describe a procedure which allows J correlation between the much better resolved IH/l5N Resonances of sequential residues, thereby bypassing the overlapping 'Ha/ I3Ca pairs. Efficient transfer of magnetization from I3Ca to I5N is possible in the present case because of the I3Ca line narrowing afforded by deuteration and 2H decoupling.
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improved 3d Triple Resonance nmr techniques applied to a 31 kda protein
Journal of Magnetic Resonance, 1992Co-Authors: Stephan Grzesiek, Ad BaxAbstract:Recently proposed 3D Triple-Resonance techniques (I-5) make it possible to obtain sequential assignment of the backbone ‘H, 15N and 13C Resonances in proteins that can be isotopically labeled with 13C and “N. Thi’s approach has been used successfully for a number of systems, including calmodulin ( 16.7 kDa) ( 1) , calmodulin complexed with a 26-residue peptide ( -20 kDa) (6), and the phospho-carrier protein IIIG’” ( 7) ( 18 kDa). All of these Triple-Resonance experiments rely on transfer of magnetization via heteronuclear one-bond Jcouplings and the sensitivity of the experiments depends strongly on the ratio of the size of the J coupling and the linewidths of the nuclei involved in the magnetization transfer process. Our original experiments were designed to minimize the number of RF pulses required for a particular pulse sequence, and thus to minimize the effect of pulse imperfections. Subsequent experience with these experiments has indicated that the effect of pulse imperfections is not as severe as originally expected, provided that pulses are properly calibrated. Hence, in order to optimize the experiments it is more important to minimize the relaxation of transverse magnetization by keeping the dephasing and rephasing delays as short as possible, especially for larger proteins. Here we demonstrate the applicability of three improved Triple-Resonance experiments to the study of interferon-y, a dimer with 134 residues per monomer and a total molecular weight of 3 1.4 kDa. Correlation of the amide proton and “N Resonance with the intraresidue 13Ca can be obtained with an experiment known as HNCA (I, 2). Note that this experiment also provides connectivity to the Ca of the preceding residue, transferring coherence via the ’ JN-Ca coupling. Because ’ JN-Ca and ’ JNxn can be of comparable magnitude (8) it may not always be possible to distinguish intrafrom interresidue connectivities with this experiment. In contrast, the HN( CO)CA experiment (4) provides correlations exclusively to the Ca Resonance of the preceding residue by relaying magnetization via the intervening carbonyl 13C spin. The HN( CO)CA experiment is closely related to the HNCO experiment, which correlates the amide ‘H and “N with the 13C of the preceding residue. In the present study of interferon-y, we employ modified versions of these experiments, using INEPT (9) instead of HMQC (10-12) type coherence transfers, and overlay the 13C15N dephasing period with a 15N evolution period
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three dimensional Triple Resonance nmr of 13c 15n enriched proteins using constant time evolution
Journal of Magnetic Resonance, 1991Co-Authors: Robert Powers, Angela M Gronenborn, Marius G Clore, Ad BaxAbstract:Recently it has been convincingly demonstrated that 3D Triple-Resonance NMR provides a practical alternative for obtaining sequential Resonance assignments in larger proteins ( I, 2). This approach requires a set of five or six 3D NMR experiments that correlate the various protein backbone nuclei. Details regarding the mechanisms and technical implementations of these experiments have been described previously (35). Two of the experiments used in this approach correlate backbone Ha and Ca Resonances with either the intraresidue carbonyl Resonance (CO) or the 15N Resonance of the succeeding residue and are referred to as HCACO and HCA ( CO) N, respectively. The present Communication describes a modification of these experiments which optimizes their sensitivity and removes the F, antiphase character of correlations. The pulse schemes of the original HCACO and HCA (CO)N experiments are very similar to the new versions that are shown in Fig. 1. The original schemes differ from the new schemes only by having a variable length evolution period of duration t, between the first pair of simultaneously applied 90” (‘H/ 13Ca) pulses and the second pair of 90” pulses, applied to “Ccu and 13C0, instead of the fixed-time duration, 2T. In both original schemes a 180” ‘H pulse is applied at the midpoint oft, to remove Ha-Ca J coupling (3). Before discussing the improved performance of the new schemes we briefly outline the basic principle of the original sequences. In both original experiments, Ha magnetization is transferred to Ca using an INEPT scheme. At the end of the t, period, Ca magnetization is transferred to the CO nucleus. In the HCACO experiment this CO magnetization evolves in the transverse plane during the second evolution period, t2, prior to being transferred back to Ca! and Ha for detection. In the HCA(CO)N experiment, Ca magnetization is also transferred to the CO nucleus, but it is subsequently relayed to “N in an HMQC manner, prior to transferring this magnetization back via CO to Ca and Ha for detection. Full details and an operator formalism description of the magnetization transfers involved have been given previously (3). To clarify the modification described in the present Communication, the pertinent product-operator formalism terms describing the magnetization transfers are briefly repeated for the HCACO experiment, with irrelevant constants omitted. Spin operators for Ha, Ca, and CO are denoted by I, A, and S, respectively. At the start of the tl evolution period, Ca magnetization is antiphase with respect to its attached Ha proton and in-phase with respect to the directly attached Cp and
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four dimensional heteronuclear Triple Resonance nmr of isotopically enriched proteins for sequential assignment of backbone atoms
Journal of Magnetic Resonance, 1991Co-Authors: Lewis E. Kay, Mitsuhiko Ikura, Guang Zhu, Ad BaxAbstract:Any two-dimensional NMR experiment that involves a relay step to transfer magnetization between two nuclei via an intermediate nucleus can be converted into a three-dimensional experiment where the third axis provides the Resonance frequency of the intermediate spin. Indeed, many of the 3D experiments proposed so far are constructed according to this principle and have popular 2D relay analogs. For example, the 3D heteronuclear NOESY-HMQC (I-3) HMQC-COSY (4), HOHAHA-HMQC (5), and ROESY-HMQC (6) experiments are very similar to their 2D counterparts ( 6-1 I ) . Similarly, the 3D homonuclear NOESY-HOHAHA / TOCSY experiments ( 12, 13) are directly related to analogous 2D experiments (14, 15). Here we show an example where a 3D relay pulse scheme is advantageously converted into a 4D experiment. Recently, it has been demonstrated that four-dimensional NMR is a feasible and very practical approach for simultaneously reducing overlap and obtaining additional information regarding NOE interactions between amide and aliphatic protons ( 16). The 4D experiment described in this paper is of a different type and can be considered the analog of a previously published 3D relay experiment, known as HCA(CO)N ( I7,18). The new 4D experiment yields the combined information that can be obtained from the 3D HCA(CO)N experiment, and a 3D experiment known as HCACO, removing ambiguities that may be present in each of the corresponding 3D spectra. The HCA(CO)N 3D spectrum correlates the Ha and Ca Resonances of one amino acid residue in a protein with the 15N Resonance of the next residue, using the carbonyl Resonance (CO) as an intermediate nucleus to transfer coherence from Ccu to “N, and vice versa. The HCACO spectrum correlates intraresidue Ha, Ca, and CO chemical shifts. Both 3D and 4D experiments require a protein that is labeled uniformly at a high level (>90%) with both “N and 13C. In the 4D experiment, named HCACON, the four frequency coordinates of a Resonance are determined by the intraresidue HCY ( F4), Ca (Pi ), and CO ( F2) shifts and the i5N shift ( F3) of the next residue. The pulse sequence of this experiment is sketched in Fig. 1. The magnetization transfer pathway that gives rise to the desired Resonances
Gaetano T Montelione - One of the best experts on this subject based on the ideXlab platform.
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a generalized approach to automated nmr peak list editing application to reduced dimensionality Triple Resonance spectra
Journal of Magnetic Resonance, 2004Co-Authors: Hunter N B Moseley, Thomas Szyperski, Gaetano T Montelione, Nadeem Riaz, James M AraminiAbstract:We present an algorithm and program called Pattern Picker that performs editing of raw peak lists derived from multidimensional NMR experiments with characteristic peak patterns. Pattern Picker detects groups of correlated peaks within peak lists from reduced dimensionality Triple Resonance (RD-TR) NMR spectra, with high fidelity and high yield. With typical quality RD-TR NMR data sets, Pattern Picker performs almost as well as human analysis, and is very robust in discriminating real peak sets from noise and other artifacts in unedited peak lists. The program uses a depth-first search algorithm with short-circuiting to efficiently explore a search tree representing every possible combination of peaks forming a group. The Pattern Picker program is particularly valuable for creating an automated peak picking/editing process. The Pattern Picker algorithm can be applied to a broad range of experiments with distinct peak patterns including RD, G-matrix Fourier transformation (GFT) NMR spectra, and experiments to measure scalar and residual dipolar coupling, thus promoting the use of experiments that are typically harder for a human to analyze. Since the complexity of peak patterns becomes a benefit rather than a drawback, Pattern Picker opens new opportunities in NMR experiment design.
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assignment validation software suite for the evaluation and presentation of protein Resonance assignment data
Journal of Biomolecular NMR, 2004Co-Authors: Hunter N B Moseley, Gaetano T Montelione, Gurmukh SahotaAbstract:We present a set of utilities and graphical user interface (GUI) tools for evaluating the quality of protein Resonance assignments. The Assignment Validation Software (AVS) suite, together with new GUI features in the AutoAssign software package, provides a set of reports and graphs for validating protein Resonance assignment data before its use in structure analysis and/or submission to the BioMagResBank (BMRB). Input includes a listing of Resonance assignments and a summary of sequential connectivity data (i.e. Triple Resonance, NOE, or other data) used in deriving the assignments. These tools are useful for evaluating the accuracy of protein Resonance assignments determined by either automated or manual methods.
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rapid analysis of protein backbone Resonance assignments using cryogenic probes a distributed linux based computing architecture and an integrated set of spectral analysis tools
Journal of Structural and Functional Genomics, 2002Co-Authors: Daniel Monleo, Thomas Szyperski, Kimberly Colso, Hunte N Moseley, Clemens Ankli, Robe E Oswald, Gaetano T MontelioneAbstract:Rapid data collection, spectral referencing, processing by time domain deconvolution, peak picking and editing, and assignment of NMR spectra are necessary components of any efficient integrated system for protein NMR structure analysis. We have developed a set of software tools designated AutoProc, AutoPeak, and AutoAssign, which function together with the data processing and peak-picking programs NMRPipe and Sparky, to provide an integrated software system for rapid analysis of protein backbone Resonance assignments. In this paper we demonstrate that these tools, together with high-sensitivity Triple Resonance NMR cryoprobes for data collection and a Linux-based computer cluster architecture, can be combined to provide nearly complete backbone Resonance assignments and secondary structures (based on chemical shift data) for a 59-residue protein in less than 30 hours of data collection and processing time. In this optimum case of a small protein providing excellent spectra, extensive backbone Resonance assignments could also be obtained using less than 6 hours of data collection and processing time. These results demonstrate the feasibility of high throughput Triple Resonance NMR for determining Resonance assignments and secondary structures of small proteins, and the potential for applying NMR in large scale structural proteomics projects.Abbreviations: BPTI – bovine pancreatic trypsin inhibitor; LP – linear prediction; FT – Fourier transform; S/N – signal-to-noise ratio; FID – free induction decay
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automated analysis of protein nmr assignments using methods from artificial intelligence
Journal of Molecular Biology, 1997Co-Authors: Diane E Zimmerman, Robert Powers, Casimir A Kulikowski, Yuanpeng Huang, Wenqing Feng, Mitsuru Tashiro, Sakurako Shimotakahara, Chen Ya Chien, Gaetano T MontelioneAbstract:An expert system for determining Resonance assignments from NMR spectra of proteins is described. Given the amino acid sequence, a two-dimensional 15N-1H heteronuclear correlation spectrum and seven to eight three-dimensional Triple-Resonance NMR spectra for seven proteins, AUTOASSIGN obtained an average of 98% of sequence-specific spin-system assignments with an error rate of less than 0.5%. Execution times on a Sparc 10 workstation varied from 16 seconds for smaller proteins with simple spectra to one to nine minutes for medium size proteins exhibiting numerous extra spin systems attributed to conformational isomerization. AUTOASSIGN combines symbolic constraint satisfaction methods with a domain-specific knowledge base to exploit the logical structure of the sequential assignment problem, the specific features of the various NMR experiments, and the expected chemical shift frequencies of different amino acids. The current implementation specializes in the analysis of data derived from the most sensitive of the currently available Triple-Resonance experiments. Potential extensions of the system for analysis of additional types of protein NMR data are also discussed.
Gerhard Wagner - One of the best experts on this subject based on the ideXlab platform.
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unambiguous assignment of nmr protein backbone signals with a time shared Triple Resonance experiment
Journal of Biomolecular NMR, 2005Co-Authors: Dominique P Frueh, Haribabu Arthanari, Gerhard WagnerAbstract:An experiment that provides a simple procedure to assign backbone nuclei in proteins is presented. The method relies on time-shared evolution of the coherences present in the (HN)NCAHA and (HA)CANNH experiments. By exploiting the fact that some of the coherences are common to both experiments the alpha and amide protons that are simultaneously detected are correlated with each other and with nitrogen and carbon nuclei. Thus, simultaneous assignment of Hα, HN, Cα and N signals is achieved in a single 3D spectrum. The experiment was tested on the streptococcal protein G B1 domain (GB1) which was easily assigned using a “stairway” procedure and on an 11 kDa domain of the yeast transcriptional co-activator Gal11.
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accelerated acquisition of high resolution Triple Resonance spectra using non uniform sampling and maximum entropy reconstruction
Journal of Magnetic Resonance, 2004Co-Authors: David Rovnyak, Jeffrey C. Hoch, Dominique P Frueh, Mallika Sastry, Zhenyu J Sun, Alan S Stern, Gerhard WagnerAbstract:Abstract Non-uniform sampling is shown to provide significant time savings in the acquisition of a suite of three-dimensional NMR experiments utilized for obtaining backbone assignments of H, N, C ′ , CA, and CB nuclei in proteins : HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB. Non-uniform sampling means that data were collected for only a subset of all incremented evolution periods, according to a user-specified sampling schedule. When the suite of six 3D experiments was acquired in a uniform fashion for an 11 kDa cytoplasmic domain of a membrane protein at 1.5 mM concentration, a total of 146 h was consumed. With non-uniform sampling, the same experiments were acquired in 32 h and, through subsequent maximum entropy reconstruction, yielded spectra of similar quality to those obtained by conventional Fourier transform of the uniformly acquired data. The experimental time saved with this methodology can significantly accelerate protein structure determination by NMR, particularly when combined with the use of automated assignment software, and enable the study of samples with poor stability at room temperature. Since it is also possible to use the time savings to acquire a greater numbers of scans to increase sensitivity while maintaining high resolution, this methodology will help extend the size limit of proteins accessible to NMR studies, and open the way to studies of samples that suffer from solubility problems.
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ibis a tool for automated sequential assignment of protein spectra from Triple Resonance experiments
Journal of Biomolecular NMR, 2003Co-Authors: Sven G Hyberts, Gerhard WagnerAbstract:We have developed a tool for computer-assisted assignments of protein NMR spectra from Triple Resonance data. The program is designed to resemble established manual assignment procedures as closely as possible. IBIS exports its results in XEASY format. Thus, using IBIS the operator has continuous visual and accounting control over the progress of the assignment procedure. IBIS achieves complete assignments for those residues that exhibit sequential Triple Resonance connectivities within a few hours or days.
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Improved resolution in Triple-Resonance spectra by nonlinear sampling in the constant-time domain
Journal of biomolecular NMR, 1994Co-Authors: Peter Schmieder, Gerhard Wagner, Alan S Stern, Jeffrey C. HochAbstract:Nonlinear sampling along the constant-time dimension is applied to the constant-time HNCO spectrum of the dimerization domain of Gal4. Nonlinear sampling was used for the nitrogen dimension, while the carbon and proton dimensions were sampled linearly. A conventional ct-HNCO spectrum is compared with a nonlinearly sampled spectrum, where the gain in experiment time obtained from nonlinear sampling is used to increase the resolution in the carbonyl dimension. Nonlinearly sampled data are processed by maximum entropy reconstruction. It is shown that the nonlinearly sampled spectrum has a higher resolution, although it was recorded in less time. The constant intensity of the signal in the constant-time dimension allows for a variety of sampling schedules. A schedule of randomly distributed sampling points yields the best results. This general method can be used to significantly increase the quality of heteronuclear constant-time spectra.
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a constant time three dimensional Triple Resonance pulse scheme to correlate intraresidue 1hn 15n and 13c chemical shifts in 15n13c labelled proteins
Journal of Magnetic Resonance, 1992Co-Authors: Venkataraman Thanabal, Robert T Clubb, Gerhard WagnerAbstract:Sequence-specif ic Resonance assignments are a prerequisite for structural and dynamical interpretation of protein NMR spectra. For proteins smaller than 10 kDa assignment strategies have relied upon through-bond correlations in homonuclear COSY and TOCSY spectra to identify Resonances associated with particular spin systems. Conformation-dependent nuclear Overhauser effects are then emp loyed to sequentially connect these spin systems (1-5). In larger proteins, however, extensive Resonance overlap and decreased sensitivity of experiments utilizing ‘H‘H scalar couplings have hindered this approach. ‘HI%15N Triple-Resonance experiments provide a conformation-independent approach for the assignment of backbone Resonances in I%15N-labeled large proteins (6-13). In addition these experiments allow accurate measurement of coupling constants in proteins with large linewidths (14, 15). These experiments exploit large heteronuclear one-bond couplings to transfer magnetization with the sensitivity of indirect detection. As demonstrated in calmodulin ( 16, 17) backbone assignments utilize four tr iple-Resonance experiments [ HNCa, HNCO, HCaCO, and HCA(CO)N] and the 3D TOCSY-HMQC experiment ( 18). A fifth tr iple-Resonance experiment [ H( CA)NHN] is a useful complement to the TOCSY-HMQC experiment, correlating ‘HN “N and ‘H” Resonances ( 7, 9, 13). Furthermore, a sixth experiment, the HN (CO ) CA. has been introduced, providing additional sequential information ( 11) . Of the experiments listed above, those which detect am ide protons [ HNCA, HNCO, H(CA)NHN, HN(CO)Ca] need to be collected in H20, while those with detection of (Y protons [ HCACO and HCA( CO)N] are best performed in 2H20. This causes difficulties if assignments are based on al ignment of C’ chemical shifts, because these Resonances may show significant isotope shifts, depending on whether the carbonyl is hydrogen bonded to a proton or a deuteron. It is therefore desirable to perform the ma jority of experiments in the same solvent ( H20), restricting oneself to those experiments which detect am ide protons [ HNCA, HNCO, HN( CO)CA. H( CA )NHN 1.
Kurt Wuthrich - One of the best experts on this subject based on the ideXlab platform.
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nmr assignment and secondary structure determination of an octameric 110 kda protein using trosy in Triple Resonance experiments
Journal of the American Chemical Society, 2000Co-Authors: Michael Salzmann, Gerhard Wider, Konstantin Pervushin, And Hans Senn, Kurt WuthrichAbstract:TROSY-type Triple Resonance experiments with the uniformly 2H,13C,15N-labeled 7,8-dihydroneopterin aldolase (DHNA) from Staphylococcus aureus, which is a symmetric homooctamer protein of molecular mass 110 kDa, showed 20-fold to 50-fold sensitivity gains when compared to the corresponding conventional Triple Resonance NMR experiments. On this basis, sequential connectivities could be established for nearly all pairs of neighboring residues in DHNA. TROSY-type nuclear Overhauser enhancement spectroscopy yielded additional data to close the remaining gaps in the sequential assignment, and provided supplementary information on the secondary structure. Complete sequence-specific assignments of the 121-residue polypeptide chain in this 110 kDa octamer could thus be obtained in aqueous solution at 20 °C, and the regular secondary structures in the solution conformation were found to coincide nearly identically with those in the crystal structure of the DHNA octamer.
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improved sensitivity and coherence selection for 15n 1h trosy elements in Triple Resonance experiments
Journal of Biomolecular NMR, 1999Co-Authors: Michael Salzmann, Gerhard Wider, Konstantin Pervushin, Kurt WuthrichAbstract:In experiments with proteins of molecular weights around 100 kDa the implementation of [15N,1H]-TROSY-elements in [15N]-constant-time Triple Resonance experiments yields sensitivity enhancements of one to two orders of magnitude. An additional gain of 10 to 20% may be obtained with the use of ‘sensitivity enhancement elements’. This paper describes a novel sensitivity enhancement scheme which is based on concatenation of the 13 Cα → 15N magnetization transfer with the ST2-PT element, and which enables proper TROSY selection of the 15N multiplet components.
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TROSY-type Triple-Resonance Experiments for Sequential NMR Assignments of Large Proteins
Journal of the American Chemical Society, 1999Co-Authors: Michael Salzmann, Gerhard Wider, Hans Senn, Konstantin Pervushin, Kurt WuthrichAbstract:Transverse relaxation-optimized spectroscopy (TROSY) was implemented in the four Triple Resonance experiments ( 15 N, 1 H)-TROSY-HN(CO)CA, ( 15 N, 1 H)-TROSY-HN(CA)CO, ( 15 N, 1 H)-TROSY-HNCACB, and ( 15 N, 1 H)-TROSY-HN(CO)CACB. Combined with ( 15 N, 1 H)-TROSY-HNCA and ( 15 N, 1 H)-TROSY-HNCO (Salzmann, M.; Pervushin, K.; Wider, G.; Senn, H. Wuthrich, K.Proc. Natl. Acad. Sci. U.S.A.1998, 13585- 13590) these experiments represent a suite of TROSY-type Triple Resonance experiments that enables sequential backbone assignment of proteins. When used with the 23 kDa 2 H/ 13 C/ 15 N-labeled protein gyrase 23B, a comparison with the corresponding conventional NMR experiments showed, on average over the entire amino acid sequence, a 3-fold sensitivity gain for each of the four experiments. The use of the TROSY principle in Triple Resonance experiments thus promises to enable Resonance assignments for significantly larger proteins than what is achievable today with the corresponding conventional NMR experiments.
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TROSY in Triple-Resonance experiments: New perspectives for sequential NMR assignment of large proteins
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Michael Salzmann, Gerhard Wider, Hans Senn, Konstantin Pervushin, Kurt WuthrichAbstract:The NMR assignment of 13C, 15N-labeled proteins with the use of Triple Resonance experiments is limited to molecular weights below ∼25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into Triple Resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.
