The Experts below are selected from a list of 11622 Experts worldwide ranked by ideXlab platform

Peter Güntert - One of the best experts on this subject based on the ideXlab platform.

  • 1 h 13 c and 15 n Resonance Assignment of the yth domain of ythdc2
    Biomolecular Nmr Assignments, 2021
    Co-Authors: Ryuta Endo, Kanako Kuwasako, Mari Takahashi, K Tsuda, Takashi Nagata, S Watanabe, Akiko Tanaka, N Kobayashi, T Kigawa, Peter Güntert
    Abstract:

    In humans, YTH (YT521-B homology) domain containing protein 2 (YTHDC2) plays a crucial role in the phase-shift from mitosis to meiosis. YTH domains bind to methylated adenosine nucleotides such as m6A. In a phylogenic tree, the YTH domain of YTHDC2 (YTH2) and that of the YTH containing protein YTHDC1 (YTH1) belong to the same sub-group. However, the binding affinity of m6A differs between these proteins. Here, we report 1H, 13C and 15N Resonance Assignment of YTH2 and its solution structure to examine the difference of the structural architecture and the dynamic properties of YTH1 and YTH2. YTH2 adopts a β1-α1-β2-α2-β3-β4-β5-α3-β6-α4 topology, which was also observed in YTH1. However, the β4-β5 loops of YTH1 and YTH2 are distinct in length and amino acid composition. Our data revealed that, unlike in YTH1, the structure of m6A-binding pocket of YTH2 formed by the β4-β5 loop is stabilized by electrostatic interaction. This Assignment and the structural information for YTH2 will provide the insight on the further functional research of YTHDC2.

  • Automated Backbone NMR Resonance Assignment of Large Proteins Using Redundant Linking from a Single Simultaneous Acquisition
    Journal of the American Chemical Society, 2020
    Co-Authors: Jan Stanek, Peter Güntert, Tobias Schubeis, Piotr Paluch, Loren Andreas, Guido Pintacuda
    Abstract:

    Thanks to magic-angle spinning (MAS) probes with frequencies of 60-100 kHz, the benefit of high sensitivity 1 H detection can now be broadly realized in biomolecular solid-state NMR for the analysis of microcrystalline, sedimented, or lipid-embedded preparations. Nonetheless, performing the Assignment of all Resonances remains a rate-limiting step in protein structural studies, and even the latest optimized protocols fail to perform this step when the protein size exceeds ~20 kDa. Here we leverage the benefits of fast (100 kHz) MAS and high (800 MHz) magnetic fields to design an approach that lifts this limitation. Through the creation, conservation and acquisition of independent magnetization pathways within a single triple-Resonance MAS NMR experiment, a single self-consistent dataset can be acquired, providing enhanced sensitivity, reduced vulnerability to machine or sample instabilities, and highly redundant linking that supports fully-automated peak picking and Resonance Assignment. The method, dubbed RAVASSA (Redundant Assignment Via A Single Simultaneous Acquisition), is demonstrated with the Assignment of the largest protein to date in the solid state, the 42.5 kDa maltose binding protein, using a single fully protonated microcrystalline sample and one week of spectrometer time.

  • automatic structure based nmr methyl Resonance Assignment in large proteins
    Nature Communications, 2019
    Co-Authors: Iva Pritisanac, Peter Güntert, Julia M Wurz, Reid T Alderson
    Abstract:

    Isotopically labeled methyl groups provide NMR probes in large, otherwise deuterated proteins. However, the Resonance Assignment constitutes a bottleneck for broader applicability of methyl-based NMR. Here, we present the automated MethylFLYA method for the Assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA is applied to five proteins (28–358 kDa) comprising a total of 708 isotope-labeled methyl groups, of which 612 contribute NOESY cross peaks. MethylFLYA confidently assigns 488 methyl groups, i.e. 80% of those with NOESY data. Of these, 459 agree with the reference, 6 were different, and 23 were without reference Assignment. MethylFLYA assigns significantly more methyl groups than alternative algorithms, has an average error rate of 1%, modest runtimes of 0.4–1.2 h, and can handle arbitrary isotope labeling patterns and data from other types of NMR spectra. The structures and dynamics of large proteins can be studied with methyl-based NMR but peak Assignment is still challenging. Here the authors present MethylFLYA that allows automated Assignment of methyl groups and apply it to five proteins with molecular weights in the range from 28 to 358 kDa.

  • automatic structure based nmr methyl Resonance Assignment in large proteins
    bioRxiv, 2019
    Co-Authors: Iva Pritisanac, Peter Güntert, Julia M Wurz, Reid T Alderson
    Abstract:

    Abstract Isotope-labeled methyl groups provide NMR probes that can be observed in very large, otherwise deuterated systems and enable investigations of protein structure, dynamics and mechanisms. However, the Assignment of Resonances to specific methyls in the protein is expensive and time-consuming, which limits the use of methyl-based NMR for large proteins. To resolve this bottleneck, methyl Assignment methods have been developed. However, these remain limited regarding complete automation, computational feasibility, and/or the extent and accuracy of the Assignments. Here, we present the automated MethylFLYA method for the Assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA was applied to five proteins (28–358 kDa) comprising a total of 708 isotope-labeled methyl groups, of which 674 had manually determined 1H/13C reference Assignments and 614 showed cross peaks in the available NOESY peak lists. MethylFLYA confidently assigned 488 methyl groups, i.e. 79% of those with NOESY data. Of these, 460 agreed with the reference, 5 were different, and 23 concerned methyls without reference Assignment. For high-quality NOESY spectra, automatic NOESY peak picking followed by Resonance Assignment with MethylFLYA can yield results comparable to those obtained from manually prepared peak lists, indicating the feasibility of unbiased, fully automatic methyl Resonance Assignment starting directly from the NMR spectra. Overall, MethylFLYA assigns significantly more methyl groups than other algorithms, has an average error rate of 1%, modest runtimes of 0.4–1.2 h, and flexibility to handle arbitrary isotope labeling patterns and include data from other types of NMR spectra.

  • structure based methyl Resonance Assignment with methylflya
    bioRxiv, 2019
    Co-Authors: Iva Pritisanac, Julia M Wurz, T R Alderson, Peter Güntert
    Abstract:

    Methyl groups provide crucial NMR probes for investigating protein structure, dynamics and mechanisms in systems that are too large for NMR with uniform isotope labeling. This requires the Assignment of methyl signals in the NMR spectra to specific methyl groups in the protein, an expensive and time-consuming endeavor that limits the use of methyl-based NMR for large proteins. To resolve this bottleneck, several methyl Resonance Assignment methods have been developed. These approaches remain limited with regard to complete automation and/or the extent and accuracy of the Assignments. Here, we present the completely automated MethylFLYA method for the Assignment of methyl groups. MethylFLYA requires as input exclusively methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. The algorithm was applied to five proteins of 28-358 kDa mass with a total of 708 isotope-labeled methyl groups. Manually made 1H/13C reference Assignments were available for 674 methyls. The available experimental peak lists contained NOESY cross peaks for 614 methyls. MethylFLYA confidently assigned 488 methyls, i.e. 79% of those with NOESY data. Of these Assignments, 460 agreed with the reference, 5 were different (and 23 concerned methyls without reference Assignment). For three proteins of 28, 81, and 358 kDa, all confident Assignments by MethylFLYA were correct. We furthermore show that, for high-quality NOESY spectra, automatic picking of NOE signals followed by Resonance Assignment with MethylFLYA can yield results that are comparable to those obtained for manually prepared peak lists, indicating the feasibility of unbiased, fully automatic methyl Resonance Assignment starting directly from the NMR spectra. This renders MethylFLYA an advantageous alternative to existing approaches for structure-based methyl Assignment. MethylFLYA assigns, for most proteins, significantly more methyl groups than other algorithms, has an average error rate of 1%, modest runtimes of 0.4-1.2 h for the five proteins, and flexibility to handle arbitrary isotope labeling patterns and include data from other types of NMR spectra.

Adam Lange - One of the best experts on this subject based on the ideXlab platform.

  • bsh cp based 3d solid state nmr experiments for protein Resonance Assignment
    Journal of Biomolecular NMR, 2014
    Co-Authors: Chaowei Shi, Birgit Habenstein, Hannes Klaus Fasshuber, Veniamin Chevelkov, Shengqi Xiang, Suresh K Vasa, Stefan Becker, Adam Lange
    Abstract:

    We have recently presented band-selective homonuclear cross-polarization (BSH-CP) as an efficient method for CO–CA transfer in deuterated as well as protonated solid proteins. Here we show how the BSH-CP CO–CA transfer block can be incorporated in a set of three-dimensional (3D) solid-state NMR (ssNMR) pulse schemes tailored for Resonance Assignment of proteins at high static magnetic fields and moderate magic-angle spinning rates. Due to the achieved excellent transfer efficiency of 33 % for BSH-CP, a complete set of 3D spectra needed for unambiguous Resonance Assignment could be rapidly recorded within 1 week for the model protein ubiquitin. Thus we expect that BSH-CP could replace the typically used CO–CA transfer schemes in well-established 3D ssNMR approaches for Resonance Assignment of solid biomolecules.

  • 13c spin dilution for simplified and complete solid state nmr Resonance Assignment of insoluble biological assemblies
    Journal of the American Chemical Society, 2011
    Co-Authors: Antoine Loquet, Stefan Becker, Guohua Lv, Karin Giller, Adam Lange
    Abstract:

    A strategy for simplified and complete Resonance Assignment of insoluble and noncrystalline proteins by solid-state NMR (ssNMR) spectroscopy is presented. Proteins produced with [1-13C]- or [2-13C]glucose are very sparsely labeled, and the resulting 2D ssNMR spectra exhibit smaller line widths (by a factor of ∼2 relative to uniformly labeled proteins) and contain a reduced number of cross-peaks. This allows for an accelerated and straightforward Resonance Assignment without the necessity of time-consuming 3D spectroscopy or sophisticated pulse sequences. The strategy aims at complete backbone and side-chain Resonance Assignments based on bidirectional sequential walks. The approach was successfully demonstrated with the de novo Assignment of the Type Three Secretion System PrgI needle protein. Using a limited set of simple 2D experiments, we report a 97% complete Resonance Assignment of the backbone and side-chain 13C atoms.

  • 13c spin dilution for simplified and complete solid state nmr Resonance Assignment of insoluble biological assemblies
    Journal of the American Chemical Society, 2011
    Co-Authors: Antoine Loquet, Stefan Becker, Karin Giller, Adam Lange
    Abstract:

    A strategy for simplified and complete Resonance Assignment of insoluble and noncrystalline proteins by solid-state NMR (ssNMR) spectroscopy is presented. Proteins produced with [1-13C]- or [2-13C]...

  • low power solid state nmr experiments for Resonance Assignment under fast magic angle spinning
    ChemPhysChem, 2009
    Co-Authors: Vinesh Vijayan, Stefan Becker, Jeanphilippe Demers, Jacek Biernat, Eckhard Mandelkow, Adam Lange
    Abstract:

    Solid-state NMR has evolved in the past decade into a powerful technique for the characterization of biomolecular structure and dynamics. Micro-crystalline globular proteins, amyloid fibrils, and membrane proteins can now be routinely studied using solid-state NMR techniques. This was made possible in part due to the development of 2D and 3D homonuclear and heteronuclear experiments that correlate C and N spins for Resonance Assignment as well as for obtaining longrange distance restraints in structure elucidation. Remarkable developments in magic-angle spinning (MAS) probe technology also contributed to this success. Now, a new generation of commercially available 1.3 mm probes can reach above 60 kHz of MAS. This allows for more efficient averaging of strong dipolar couplings, hence providing better resolution in highly crowded protein spectra. On the other hand, fast spinning reduces the effectiveness of many of the routinely used NMR experiments for obtaining Resonance Assignments. For example, at low MAS ( 15 kHz), C C correlations are often measured by proton-driven spin diffusion (PDSD). Under very fast MAS, efficient averaging of dipolar couplings renders PDSD experiments ineffective. Instead, selective dipolar recoupling of spins becomes necessary to allow for efficient polarization transfer. Herein, we introduce a complete set of low-power solidstate NMR experiments sufficient for protein Resonance Assignment under fast MAS (>60 kHz), including sequential N C correlation experiments. The low rf (radio frequency)-field requirements of our experiments prevent considerable heating of the sample, thus avoiding protein degradation and making this approach well-suited for the investigation of temperaturesensitive biomolecules. As an application, NCA, N(CO)CA, and C C correlation spectra were recorded at 60 kHz MAS on less than 1 mg of [C, N] isotope-labeled sample. We also demonstrate that our approach can be readily performed on protein samples in which the H T1 relaxation times are shortened by means of paramagnetic doping. Here, the reduced recycle delay enhances the sensitivity but requires the use of NMR sequences with low-power deposition, as described herein. Figure 1 presents the different pulse schemes that were used to obtain N C and C C correlations. At low MAS, the initial cross-polarization (CP) transfer from protons to low-g nuclei generally requires high power irradiation on both channels. In contrast, under fast MAS, efficient CP transfer is also possible at low rf fields. Our pulse schemes use second-order cross-polarization (SOCP) for the initial magnetization transfer. SOCP at the n=0 Hartman–Hahn condition relies on second-order crossterms between homonuclear and heteronuclear couplings. SOCP works efficiently at low rf fields if sufficient care is taken to avoid detrimental dipolar and/or CSA recoupling conditions at the used rf-field amplitudes. We employed rf fields of 9 kHz—well below all Resonance conditions. SOCP is intrinsically band-selective as only weak rf fields are applied. The rf fields employed here are sufficient to excite all N protein backbone

Beat H. Meier - One of the best experts on this subject based on the ideXlab platform.

  • solid state 13c 15n nmr Resonance Assignment of hepatitis b virus core protein
    Biomolecular Nmr Assignments, 2018
    Co-Authors: Lauriane Lecoq, Beat H. Meier, Shishan Wang, Thomas Wiegand, Stephane Bressanelli, Michael Nassal, Anja Bockmann
    Abstract:

    Each year, nearly 900,000 deaths are due to serious liver diseases caused by chronic hepatitis B virus infection. The viral particle is composed of an outer envelope and an inner icosahedral nucleocapsid formed by multiple dimers of a ~ 20 kDa self-assembling core protein (Cp). Here we report the solid-state 13C and 15N Resonance Assignments of the assembly domain, Cp149, of the core protein in its capsid form. A secondary chemical shift analysis of the 140 visible residues suggests an overall alpha-helical three-dimensional fold matching that derived for Cp149 from the X-ray crystallography of the capsid, and from solution-state NMR of the Cp149 dimer. Interestingly, however, at three distinct regions the chemical shifts in solution differ significantly between core proteins in the capsid state versus in the dimer state, strongly suggesting the respective residues to be involved in capsid assembly.

  • protein Resonance Assignment at mas frequencies approaching 100 khz a quantitative comparison of j coupling and dipolar coupling based transfer methods
    Journal of Biomolecular NMR, 2015
    Co-Authors: Susanne Penzel, Matthias Ernst, Albert A Smith, Vipin Agarwal, Andreas Hunkeler, Mailiis Org, Ago Samoson, Anja Bockmann, Beat H. Meier
    Abstract:

    We discuss the optimum experimental conditions to obtain Assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100 kHz. We present a systematic examination of the MAS dependence of the amide proton T 2′ times and a site-specific comparison of T 2′ at 93 kHz versus 60 kHz MAS frequency. A quantitative analysis of transfer efficiencies of building blocks, as they are used for typical 3D experiments, was performed. To do this, we compared dipolar-coupling and J-coupling based transfer steps. The building blocks were then combined into 3D experiments for sequential Resonance Assignment, where we evaluated signal-to-noise ratio and information content of the different 3D spectra in order to identify the best Assignment strategy. Based on this comparison, six experiments were selected to optimally assign the model protein ubiquitin, solely using spectra acquired at 93 kHz MAS. Within 3 days of instrument time, the required spectra were recorded from which the backbone Resonances have been assigned to over 96 %.

  • Automated solid-state NMR Resonance Assignment of protein microcrystals and amyloids
    Journal of Biomolecular NMR, 2013
    Co-Authors: Elena Schmidt, Beat H. Meier, Anja Bockmann, Julia Gath, Birgit Habenstein, Francesco Ravotti, Kathrin Székely, Matthias Huber, Lena Buchner, Peter Güntert
    Abstract:

    Solid-state NMR is an emerging structure determination technique for crystalline and non-crystalline protein assemblies, e.g., amyloids. Resonance Assignment constitutes the first and often very time-consuming step to a structure. We present ssFLYA, a generally applicable algorithm for automatic Assignment of protein solid-state NMR spectra. Application to microcrystals of ubiquitin and the Ure2 prion C-terminal domain, as well as amyloids of HET-s(218–289) and α-synuclein yielded 88–97 % correctness for the backbone and side-chain Assignments that are classified as self-consistent by the algorithm, and 77–90 % correctness if also Assignments classified as tentative by the algorithm are included.

  • ^13C, ^15N Resonance Assignment of Parts of the HET-s Prion Protein in its Amyloid Form
    Journal of Biomolecular NMR, 2006
    Co-Authors: Ansgar B. Siemer, Matthias Ernst, Christiane Ritter, Michel O. Steinmetz, Roland Riek, Beat H. Meier
    Abstract:

    The partial ^15N and ^13C solid-state NMR Resonance Assignment of the HET-s prion protein fragment 218–289 in its amyloid form is presented. It is based on experiments measured at MAS frequencies in the range of 20–40 kHz using exclusively adiabatic polarization-transfer schemes. The Resonance Assignment within each residue is based on two-dimensional ^13C––^13C correlation spectra utilizing the DREAM mixing scheme. The sequential linking of the assigned residues used a set of two- and three-dimensional ^15N––^13C correlation experiments. Almost all cross peaks visible in the spectra are assigned, but only Resonances from 43 of the 78 amino-acid residues could be detected. The missing residues are thought to be highly disordered and/or highly dynamic giving rise to broad Resonance lines that escaped detection in the experiments applied. The line widths of the observed Resonances are narrow and comparable to line widths observed in micro-crystalline samples. The 43 assigned residues are located in two fragments of about 20 residues.

  • Methods for sequential Resonance Assignment in solid, uniformly 13C, 15N labelled peptides: Quantification and application to antamanide
    Journal of Biomolecular NMR, 2001
    Co-Authors: Andreas Detken, Edme H. Hardy, Matthias Ernst, Masatsune Kainosho, Toru Kawakami, Saburo Aimoto, Beat H. Meier
    Abstract:

    The application of adiabatic polarization-transfer experiments to Resonance Assignment in solid, uniformly ^13C-^15N-labelled polypeptides is demonstrated for the cyclic decapeptide antamanide. A homonuclear correlation experiment employing the DREAM sequence for adiabatic dipolar transfer yields a complete Assignment of the C^α and aliphatic side-chain ^13C Resonances to amino acid types. The same information can be obtained from a TOBSY experiment using the recently introduced P9^1 _12 TOBSY sequence, which employs the J couplings as a transfer mechanism. A comparison of the two methods is presented. Except for some aromatic phenylalanine Resonances, a complete sequence-specific Assignment of the ^13C and ^15N Resonances in antamanide is achieved by a series of selective or broadband adiabatic triple-Resonance experiments. Heteronuclear transfer by adiabatic-passage Hartmann–Hahn cross polarization is combined with adiabatic homonuclear transfer by the DREAM and rotational-Resonance tickling sequences into two- and three-dimensional experiments. The performance of these experiments is evaluated quantitatively.

Bernhard Brutscher - One of the best experts on this subject based on the ideXlab platform.

  • BEST-TROSY experiments for time-efficient sequential Resonance Assignment of large disordered proteins
    Journal of Biomolecular NMR, 2013
    Co-Authors: Zsofia Solyom, Dieter Willbold, Melanie Schwarten, Leonhard Geist, Robert Konrat, Bernhard Brutscher
    Abstract:

    The characterization of the conformational properties of intrinsically disordered proteins (IDPs), and their interaction modes with physiological partners has recently become a major research topic for understanding biological function on the molecular level. Although multidimensional NMR spectroscopy is the technique of choice for the study of IDPs at atomic resolution, the intrinsically low resolution, and the large peak intensity variations often observed in NMR spectra of IDPs call for resolution- and sensitivity-optimized pulse schemes. We present here a set of amide proton-detected 3D BEST-TROSY correlation experiments that yield the required sensitivity and spectral resolution for time-efficient sequential Resonance Assignment of large IDPs. In addition, we introduce two proline-edited 2D experiments that allow unambiguous identification of residues adjacent to proline that is one of the most abundant amino acids in IDPs. The performance of these experiments, and the advantages of BEST-TROSY pulse schemes are discussed and illustrated for two IDPs of similar length (~270 residues) but with different conformational sampling properties.

  • ihadamac a complementary tool for sequential Resonance Assignment of globular and highly disordered proteins
    Journal of Magnetic Resonance, 2012
    Co-Authors: Sophie Feuerstein, Bernhard Brutscher, Michael J Plevin, Dieter Willbold
    Abstract:

    An experiment, iHADAMAC, is presented that yields information on the amino-acid type of individual residues in a protein by editing the (1)H-(15)N correlations into seven different 2D spectra, each corresponding to a different class of amino-acid types. Amino-acid type discrimination is realized via a Hadamard encoding scheme based on four different spin manipulations as recently introduced in the context of the sequential HADAMAC experiment. Both sequential and intra-residue HADAMAC experiments yield highly complementary information that greatly facilitate Resonance Assignment of proteins with high frequency degeneracy, as demonstrated here for a 188-residue intrinsically disordered protein fragment of the hepatitis C virus protein NS5A.

  • 1h 13c and 15n Resonance Assignment of a 179 residue fragment of hepatitis c virus non structural protein 5a
    Biomolecular Nmr Assignments, 2011
    Co-Authors: Bernhard Brutscher, Sophie Feuerstein, Dieter Willbold, Zsofia Solyom, Amine Aladag, Silke Hoffmann
    Abstract:

    Non-structural protein 5A (NS5A) plays an important role in the life cycle of hepatitis C virus. This proline-rich phosphoprotein is organized into three domains. Besides its role in virus replication and virus assembly, NS5A is involved in a variety of cellular regulation processes. Recent studies on domain 2 and 3 revealed that both belong to the class of intrinsically disordered proteins as they adopt a natively unfolded state. In particular, domain 2 together with its vicinal regions is responsible for NS5A’s multiple interactions with other proteins necessary for virus persistence. The low chemical shift dispersion observed for instrinsically disordered proteins presents a challenge for NMR spectroscopy. Here we report sequential Resonance Assignment of a 179-residue fragment of NS5A, comprising the entire domain 2, using a set of sensitivity and resolution optimized 3D correlation experiments, as well as amino-acid-type editing in 1H-15N correlation spectra. Our Assignment reveals the presence of several segments with high propensity to form α-helical structure that may be of importance to the function of this protein fragment as a versatile interaction platform.

  • Highly automated protein backbone Resonance Assignment within a few hours: the «BATCH» strategy and software package
    Journal of Biomolecular NMR, 2009
    Co-Authors: Ewen Lescop, Bernhard Brutscher
    Abstract:

    Sequential Resonance Assignment represents an essential step towards the investigation of protein structure, dynamics, and interaction surfaces. Although the experimental sensitivity has significantly increased in recent years, with the availability of high field magnets and cryogenically cooled probes, Resonance Assignment, even of small globular proteins, still generally requires several days of data collection and analysis using standard protocols. Here we introduce the BATCH strategy for fast and highly automated backbone Resonance Assignment of ^13C, ^15N-labelled proteins. BATCH makes use of the fast data acquisition and analysis tools BEST, ASCOM, COBRA, and HADAMAC, recently developed in our laboratory. An improved Hadamard encoding scheme, presented here, further increases the performance of the HADAMAC experiment. A new software platform, interfaced to the NMRView software package, has been developed that enables highly automated NMR data processing and analysis, sequential Resonance Assignment, and ^13C chemical shift extraction. We demonstrate for four small globular proteins that sequential Resonance Assignment can be routinely obtained within a few hours, or less, in a highly automated and robust way.

  • hadamard amino acid type edited nmr experiment for fast protein Resonance Assignment
    Journal of the American Chemical Society, 2008
    Co-Authors: Ewen Lescop, Rodolfo M Rasia, Bernhard Brutscher
    Abstract:

    An original Hadamard-encoding scheme allows discrimination among seven amino acid types in a single two-dimensional NMR experiment. Combined with hyperdimensional NMR techniques, this presents a promising new method for fast, automated backbone Resonance Assignment of proteins in only a few hours time.

Stefan Becker - One of the best experts on this subject based on the ideXlab platform.

  • backbone and side chain Resonance Assignment of the a147t polymorph of mouse tspo in complex with a high affinity radioligand
    Biomolecular Nmr Assignments, 2016
    Co-Authors: Mariusz Jaremko, Stefan Becker, Karin Giller, łukasz Jaremko, Markus Zweckstetter
    Abstract:

    The integral polytopic membrane protein TSPO is the target for numerous endogenous and synthetic ligands. However, the affinity of many ligands is influenced by a common polymorphism in TSPO, in which an alanine at position 147 is replaced by threonine, thereby complicating the use of several radioligands for clinical diagnosis. In contrast, the best-characterized TSPO ligand (R)-PK11195 binds with similar affinity to both variants of mitochondrial TSPO (wild-type and A147T variant). Here we report the (1)H, (13)C, (15)N backbone and side-chain Resonance Assignment of the A147T polymorph of TSPO from Mus Musculus in complex with (R)-PK11195 in DPC detergent micelles. More than 90 % of all Resonances were sequence-specifically assigned, demonstrating the ability to obtain high-quality spectral data for both the backbone and the side-chains of medically relevant integral membrane proteins.

  • bsh cp based 3d solid state nmr experiments for protein Resonance Assignment
    Journal of Biomolecular NMR, 2014
    Co-Authors: Chaowei Shi, Birgit Habenstein, Hannes Klaus Fasshuber, Veniamin Chevelkov, Shengqi Xiang, Suresh K Vasa, Stefan Becker, Adam Lange
    Abstract:

    We have recently presented band-selective homonuclear cross-polarization (BSH-CP) as an efficient method for CO–CA transfer in deuterated as well as protonated solid proteins. Here we show how the BSH-CP CO–CA transfer block can be incorporated in a set of three-dimensional (3D) solid-state NMR (ssNMR) pulse schemes tailored for Resonance Assignment of proteins at high static magnetic fields and moderate magic-angle spinning rates. Due to the achieved excellent transfer efficiency of 33 % for BSH-CP, a complete set of 3D spectra needed for unambiguous Resonance Assignment could be rapidly recorded within 1 week for the model protein ubiquitin. Thus we expect that BSH-CP could replace the typically used CO–CA transfer schemes in well-established 3D ssNMR approaches for Resonance Assignment of solid biomolecules.

  • 13c spin dilution for simplified and complete solid state nmr Resonance Assignment of insoluble biological assemblies
    Journal of the American Chemical Society, 2011
    Co-Authors: Antoine Loquet, Stefan Becker, Guohua Lv, Karin Giller, Adam Lange
    Abstract:

    A strategy for simplified and complete Resonance Assignment of insoluble and noncrystalline proteins by solid-state NMR (ssNMR) spectroscopy is presented. Proteins produced with [1-13C]- or [2-13C]glucose are very sparsely labeled, and the resulting 2D ssNMR spectra exhibit smaller line widths (by a factor of ∼2 relative to uniformly labeled proteins) and contain a reduced number of cross-peaks. This allows for an accelerated and straightforward Resonance Assignment without the necessity of time-consuming 3D spectroscopy or sophisticated pulse sequences. The strategy aims at complete backbone and side-chain Resonance Assignments based on bidirectional sequential walks. The approach was successfully demonstrated with the de novo Assignment of the Type Three Secretion System PrgI needle protein. Using a limited set of simple 2D experiments, we report a 97% complete Resonance Assignment of the backbone and side-chain 13C atoms.

  • 13c spin dilution for simplified and complete solid state nmr Resonance Assignment of insoluble biological assemblies
    Journal of the American Chemical Society, 2011
    Co-Authors: Antoine Loquet, Stefan Becker, Karin Giller, Adam Lange
    Abstract:

    A strategy for simplified and complete Resonance Assignment of insoluble and noncrystalline proteins by solid-state NMR (ssNMR) spectroscopy is presented. Proteins produced with [1-13C]- or [2-13C]...

  • low power solid state nmr experiments for Resonance Assignment under fast magic angle spinning
    ChemPhysChem, 2009
    Co-Authors: Vinesh Vijayan, Stefan Becker, Jeanphilippe Demers, Jacek Biernat, Eckhard Mandelkow, Adam Lange
    Abstract:

    Solid-state NMR has evolved in the past decade into a powerful technique for the characterization of biomolecular structure and dynamics. Micro-crystalline globular proteins, amyloid fibrils, and membrane proteins can now be routinely studied using solid-state NMR techniques. This was made possible in part due to the development of 2D and 3D homonuclear and heteronuclear experiments that correlate C and N spins for Resonance Assignment as well as for obtaining longrange distance restraints in structure elucidation. Remarkable developments in magic-angle spinning (MAS) probe technology also contributed to this success. Now, a new generation of commercially available 1.3 mm probes can reach above 60 kHz of MAS. This allows for more efficient averaging of strong dipolar couplings, hence providing better resolution in highly crowded protein spectra. On the other hand, fast spinning reduces the effectiveness of many of the routinely used NMR experiments for obtaining Resonance Assignments. For example, at low MAS ( 15 kHz), C C correlations are often measured by proton-driven spin diffusion (PDSD). Under very fast MAS, efficient averaging of dipolar couplings renders PDSD experiments ineffective. Instead, selective dipolar recoupling of spins becomes necessary to allow for efficient polarization transfer. Herein, we introduce a complete set of low-power solidstate NMR experiments sufficient for protein Resonance Assignment under fast MAS (>60 kHz), including sequential N C correlation experiments. The low rf (radio frequency)-field requirements of our experiments prevent considerable heating of the sample, thus avoiding protein degradation and making this approach well-suited for the investigation of temperaturesensitive biomolecules. As an application, NCA, N(CO)CA, and C C correlation spectra were recorded at 60 kHz MAS on less than 1 mg of [C, N] isotope-labeled sample. We also demonstrate that our approach can be readily performed on protein samples in which the H T1 relaxation times are shortened by means of paramagnetic doping. Here, the reduced recycle delay enhances the sensitivity but requires the use of NMR sequences with low-power deposition, as described herein. Figure 1 presents the different pulse schemes that were used to obtain N C and C C correlations. At low MAS, the initial cross-polarization (CP) transfer from protons to low-g nuclei generally requires high power irradiation on both channels. In contrast, under fast MAS, efficient CP transfer is also possible at low rf fields. Our pulse schemes use second-order cross-polarization (SOCP) for the initial magnetization transfer. SOCP at the n=0 Hartman–Hahn condition relies on second-order crossterms between homonuclear and heteronuclear couplings. SOCP works efficiently at low rf fields if sufficient care is taken to avoid detrimental dipolar and/or CSA recoupling conditions at the used rf-field amplitudes. We employed rf fields of 9 kHz—well below all Resonance conditions. SOCP is intrinsically band-selective as only weak rf fields are applied. The rf fields employed here are sufficient to excite all N protein backbone