Publications by authors named "Bernhard Brutscher"

77 Publications

Disentangling Chromophore States in a Reversibly Switchable Green Fluorescent Protein: Mechanistic Insights from NMR Spectroscopy.

J Am Chem Soc 2021 May 9;143(19):7521-7530. Epub 2021 May 9.

Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 38000 Grenoble, France.

The photophysical properties of fluorescent proteins, including phototransformable variants used in advanced microscopy applications, are influenced by the environmental conditions in which they are expressed and used. Rational design of improved fluorescent protein markers requires a better understanding of these environmental effects. We demonstrate here that solution NMR spectroscopy can detect subtle changes in the chemical structure, conformation, and dynamics of the photoactive chromophore moiety with atomic resolution, providing such mechanistic information. Studying rsFolder, a reversibly switchable green fluorescent protein, we have identified four distinct configurations of its -HBI chromophore, corresponding to the and isomers, with each one either protonated (neutral) or deprotonated (anionic) at the benzylidene ring. The relative populations and interconversion kinetics of these chromophore species depend on sample pH and buffer composition that alter in a complex way the strength of H-bonds that contribute in stabilizing the chromophore within the protein scaffold. We show in particular the important role of histidine-149 in stabilizing the neutral chromophore at intermediate pH values, leading to ground-state isomerization with a peculiar pH dependence. We discuss the potential implications of our findings on the pH dependence of the photoswitching contrast, a critical parameter in nanoscopy applications.
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http://dx.doi.org/10.1021/jacs.1c02442DOI Listing
May 2021

Raw nuclear magnetic resonance data of human linker histone H1x, lacking the C-terminal domain (NGH1x), and trajectory data of nanosecond molecular dynamics simulations of GH1x- and NGH1x-chromatosomes.

Data Brief 2020 Aug 16;31:105865. Epub 2020 Jun 16.

University of Johannesburg, South Africa.

Linker histone H1 plays a vital role in the packaging of DNA. H1 has a tripartite structure: a conserved central globular domain that adopts a winged-helix fold, flanked by highly variable and intrinsically unstructured N- and C-terminal domains. The datasets presented in this article include raw 2D and 3D BEST-TROSY NMR data [1H-15 N HSQC; 15 N and 13C HNCO, HN(CO)CACB, HNCACB, HN(CA)CO] recorded for NGH1x, a truncated version of H1x containing the N-terminal and globular domains, but lacking the C-terminal domain. Experiments were conducted on double-labelled (15 N and 13C) NGH1x in 'low' and 'high salt,' to investigate the secondary structure content of the N-terminal domain of H1x under these conditions. We provide modelled structures of NGH1x (in low and high salt) based on the assigned chemical shifts in PDB format. The high salt structure of NGH1x (globular domain of H1x [GH1x; PDB: 2LSO] with the H1x NTD) was docked to the nucleosome to generate NGH1x- and GH1x-chromatosomes. The GH1x-chromatosome was generated for comparative purposes to elucidate the role of the N-terminal domain. We present raw data trajectories of molecular dynamics simulations of these chromatosomes in this article. The MD dataset provides nanosecond resolution data on the dynamics of GH1x- vs NGH1x-chromatosomes, which is useful to elucidate the DNA binding properties of the N-terminal domain of H1x in chromatin, as well as the dynamic behaviour of linker DNA in these chromatosomes.
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http://dx.doi.org/10.1016/j.dib.2020.105865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334387PMC
August 2020

Spectral editing of intra- and inter-chain methyl-methyl NOEs in protein complexes.

J Biomol NMR 2020 Jan 2;74(1):83-94. Epub 2020 Jan 2.

Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS), 71, Avenue des Martyrs, 38044, Grenoble, France.

Specific isotopic labeling of methyl groups in a perdeuterated protein background enables the detection of long range NOEs in proteins or high molecular weight complexes. We introduce here an approach, combining an optimized isotopic labeling scheme with a specifically tailored NMR pulse sequence, to distinguish between intramolecular and intermolecular NOE connectivities. In hetero-oligomeric complexes, this strategy enables sign encoding of intra-subunit and inter-subunit NOEs. For homo-oligomeric assemblies, our strategy allows the specific detection of intra-chain NOEs in high resolution 3D NOESY spectra. The general principles, possibilities and limitations of this approach are presented. Applications of this approach for the detection of intermolecular NOEs in a hetero-hexamer, and the assignment of methyl H and C resonances in a homo-tetrameric protein complex are shown.
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http://dx.doi.org/10.1007/s10858-019-00293-xDOI Listing
January 2020

NMR Reveals Light-Induced Changes in the Dynamics of a Photoswitchable Fluorescent Protein.

Biophys J 2019 12 2;117(11):2087-2100. Epub 2019 Nov 2.

Institut de Biologie Structurale, Université Grenoble Alpes, CEA, CNRS, Grenoble, France. Electronic address:

The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essential. At present, little is known about the conformational dynamics of fluorescent proteins at physiological temperature and how these dynamics contribute to the observed phototransformation properties. Here, we apply high-resolution NMR spectroscopy in solution combined with in situ sample illumination at different wavelengths to investigate the conformational dynamics of rsFolder, a GFP-derived protein that can be reversibly switched between a green fluorescent state and a nonfluorescent state. Our results add a dynamic view to the static structures obtained by x-ray crystallography. Including a custom-tailored NMR toolbox in fluorescent protein research provides new opportunities for investigating the effect of mutations or changes in the environmental conditions on the conformational dynamics of phototransformable fluorescent proteins and their correlation with the observed photochemical and photophysical properties.
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http://dx.doi.org/10.1016/j.bpj.2019.10.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895687PMC
December 2019

NMRlib: user-friendly pulse sequence tools for Bruker NMR spectrometers.

J Biomol NMR 2019 May 10;73(5):199-211. Epub 2019 May 10.

Université Grenoble Alpes, CEA, CNRS, IBS, 38000, Grenoble, France.

We present NMRlib, a suite of jython-based tools designed for Bruker spectrometers (TopSpin versions 3.2-4.0) that allow easy setup, management, and exchange of NMR experiments. A NMR experiment can be set up and executed in a few clicks by navigating through the NMRlib GUI tree structure, without any further parameter adjustment. NMRlib is magnetic-field independent, and thus particularly helpful for laboratories operating multiple NMR spectrometers. NMRlib is easily personalized by adding, deleting, or reorganizing experiments. Additional tools are provided for data processing, visualization, and analysis. In particular, NMRlib contains all the polarization-enhanced fast-pulsing NMR experiments (SOFAST, BEST, HADAMAC,…) developed in our laboratory over the last decade. We also discuss some specific features that have been implemented to make these experiments most efficient and user friendly.
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http://dx.doi.org/10.1007/s10858-019-00249-1DOI Listing
May 2019

NMR assignments of human linker histone H1x N-terminal domain and globular domain in the presence and absence of perchlorate.

Biomol NMR Assign 2019 04 13;13(1):249-254. Epub 2019 Mar 13.

University of Johannesburg, PO Box 524, Auckland Park, Johannesburg, South Africa.

Human linker histone H1 plays a seminal role in eukaryotic DNA packaging. H1 has a tripartite structure consisting of a central, conserved globular domain, which adopts a winged-helix fold, flanked by two variable N- and C-terminal domains. Here we present the backbone resonance assignments of the N-terminal domain and globular domain of human linker histone H1x in the presence and absence of the secondary structure stabilizer sodium perchlorate. Analysis of chemical shift changes between the two conditions is consistent with induction of transient secondary structural elements in the N-terminal domain of H1x in high ionic strength, which suggests that the N-terminal domain adopts significant alpha-helical conformations in the presence of DNA.
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http://dx.doi.org/10.1007/s12104-019-09886-xDOI Listing
April 2019

Aromatic SOFAST-HMBC for proteins at natural C abundance.

J Magn Reson 2019 03 23;300:95-102. Epub 2019 Jan 23.

Univ. Grenoble Alpes, CEA, CNRS, IBS, 38000 Grenoble, France. Electronic address:

We propose here SOFAST-HMBC as a new complementary NMR tool for aromatic side chain assignment of protein samples at natural C abundance. The characteristic peak patterns detected in SOFAST-HMBC for each aromatic side chain allow straightforward assignment of all protons and carbons (including quaternary ones) of the aromatic ring, and for tyrosine and phenylalanine, connection to the CB of the aliphatic chain. The performance of SOFAST-HMBC is demonstrated for three small proteins (7-14 kDa) at millimolar sample concentration using modern high-field NMR instruments equipped with cryogenically cooled probes. Despite the low amount of NMR-active C nuclei in these samples, H-C multiple-bond correlation spectra of good quality were obtained in reasonable experimental times of typically less than 24 h.
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http://dx.doi.org/10.1016/j.jmr.2019.01.009DOI Listing
March 2019

BEST and SOFAST experiments for resonance assignment of histidine and tyrosine side chains in C/N labeled proteins.

J Biomol NMR 2018 Dec 21;72(3-4):115-124. Epub 2018 Nov 21.

Univ. Grenoble Alpes, CEA, CNRS, IBS, 38000, Grenoble, France.

Aromatic amino-acid side chains are essential components for the structure and function of proteins. We present herein a set of NMR experiments for time-efficient resonance assignment of histidine and tyrosine side chains in uniformly C/N-labeled proteins. The use of band-selective C pulses allows to deal with linear chains of coupled spins, thus avoiding signal loss that occurs in branched spin systems during coherence transfer. Furthermore, our pulse schemes make use of longitudinal H relaxation enhancement, Ernst-angle excitation, and simultaneous detection of H and C steady-state polarization to achieve significant signal enhancements.
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http://dx.doi.org/10.1007/s10858-018-0216-zDOI Listing
December 2018

How Detergent Impacts Membrane Proteins: Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine.

J Phys Chem Lett 2018 Mar 8;9(5):933-938. Epub 2018 Feb 8.

Université Grenoble Alpes, CNRS, CEA, IBS , 38000 Grenoble, France.

Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent-membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in DPC, they systematically correspond to nonfunctional states.
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http://dx.doi.org/10.1021/acs.jpclett.8b00269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834942PMC
March 2018

Optimized fast mixing device for real-time NMR applications.

J Magn Reson 2017 08 31;281:125-129. Epub 2017 May 31.

Institut de Biologie Structurale, Université Grenoble Alpes, CEA, CNRS, 71 avenue des Martyrs, 38044 Grenoble Cedex 9, France. Electronic address:

We present an improved fast mixing device based on the rapid mixing of two solutions inside the NMR probe, as originally proposed by Hore and coworkers (J. Am. Chem. Soc. 125 (2003) 12484-12492). Such a device is important for off-equilibrium studies of molecular kinetics by multidimensional real-time NMR spectrsocopy. The novelty of this device is that it allows removing the injector from the NMR detection volume after mixing, and thus provides good magnetic field homogeneity independently of the initial sample volume placed in the NMR probe. The apparatus is simple to build, inexpensive, and can be used without any hardware modification on any type of liquid-state NMR spectrometer. We demonstrate the performance of our fast mixing device in terms of improved magnetic field homogeneity, and show an application to the study of protein folding and the structural characterization of transiently populated folding intermediates.
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http://dx.doi.org/10.1016/j.jmr.2017.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542027PMC
August 2017

Fragment-Based NMR Study of the Conformational Dynamics in the bHLH Transcription Factor Ascl1.

Biophys J 2017 Apr;112(7):1366-1373

Institut de Biologie Structurale, Université Grenoble, Grenoble, France; Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Grenoble, France; Centre National de la Recherche Scientifique (CNRS), Grenoble, France. Electronic address:

The Achaete-scute homolog 1 (Ascl1) protein regulates a large subset of genes that leads neuronal progenitor cells to distinctive differentiation pathways during human brain development. Although it is well known that Ascl1 binds DNA as a homo- or heterodimer via its basic helix-loop-helix (bHLH) motif, little is known about the conformational sampling properties of the DNA-free full-length protein, and in particular about the bHLH domain-flanking N- and C-terminal segments, which are predicted to be highly disordered in solution. The structural heterogeneity, low solubility, and high aggregation propensity of Ascl1 in aqueous buffer solutions make high-resolution studies of this protein a challenging task. Here, we have adopted a fragment-based strategy that allowed us to obtain high-quality NMR data providing, to our knowledge, the first comprehensive high-resolution information on the structural propensities and conformational dynamics of Ascl1. The emerging picture is that of an overall extended and highly dynamic polypeptide chain comprising three helical segments and lacking persistent long-range interactions. We also show that the C-terminal helix of the bHLH domain is involved in intermolecular interactions, even in the absence of DNA. Our results contribute to a better understanding of the mechanisms of action that govern the regulation of proneural transcription factors.
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http://dx.doi.org/10.1016/j.bpj.2017.02.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390047PMC
April 2017

RNA binding and chaperone activity of the E. coli cold-shock protein CspA.

Nucleic Acids Res 2017 04;45(7):4255-4268

Institut de Biologie Structurale, Université Grenoble 1, 71 avenue des Martyrs, 38044 Grenoble Cedex 9, France.

Ensuring the correct folding of RNA molecules in the cell is of major importance for a large variety of biological functions. Therefore, chaperone proteins that assist RNA in adopting their functionally active states are abundant in all living organisms. An important feature of RNA chaperone proteins is that they do not require an external energy source to perform their activity, and that they interact transiently and non-specifically with their RNA targets. So far, little is known about the mechanistic details of the RNA chaperone activity of these proteins. Prominent examples of RNA chaperones are bacterial cold shock proteins (Csp) that have been reported to bind single-stranded RNA and DNA. Here, we have used advanced NMR spectroscopy techniques to investigate at atomic resolution the RNA-melting activity of CspA, the major cold shock protein of Escherichia coli, upon binding to different RNA hairpins. Real-time NMR provides detailed information on the folding kinetics and folding pathways. Finally, comparison of wild-type CspA with single-point mutants and small peptides yields insights into the complementary roles of aromatic and positively charged amino-acid side chains for the RNA chaperone activity of the protein.
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http://dx.doi.org/10.1093/nar/gkx044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397153PMC
April 2017

Probing Conformational Exchange Dynamics in a Short-Lived Protein Folding Intermediate by Real-Time Relaxation-Dispersion NMR.

J Am Chem Soc 2017 01 11;139(3):1065-1068. Epub 2017 Jan 11.

Institut de Biologie Structurale, Université Grenoble Alpes , 71 Avenue des Martyrs, 38044 Grenoble Cedex 9, France.

NMR spectroscopy is a powerful tool for studying molecular dynamics at atomic resolution simultaneously for a large number of nuclear sites. In this communication, we combine two powerful NMR techniques, relaxation-dispersion NMR and real-time NMR, in order to obtain unprecedented information on the conformational exchange dynamics present in short-lived excited protein states, such as those transiently accumulated during protein folding. We demonstrate the feasibility of the approach for the amyloidogenic protein β2-microglobulin that folds via an intermediate state which is believed to be responsible for the onset of the aggregation process leading to amyloid formation.
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http://dx.doi.org/10.1021/jacs.6b12089DOI Listing
January 2017

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation.

Biophys J 2015 Oct;109(7):1483-96

Institut de Biologie Structurale, Université Grenoble 1, Grenoble, France; Commissariat à l'Energie Atomique et aux Energies Alternatives, Grenoble, France; Centre National de Recherche Scientifique, Grenoble, France. Electronic address:

Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein's interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191-447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.
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http://dx.doi.org/10.1016/j.bpj.2015.06.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601004PMC
October 2015

NMR Methods for the Study of Instrinsically Disordered Proteins Structure, Dynamics, and Interactions: General Overview and Practical Guidelines.

Adv Exp Med Biol 2015 ;870:49-122

Institut de Biologie Structurale, Université Grenoble 1, CNRS, CEA, 71 avenue des Martyrs, 38044, Grenoble Cedex 9, France.

Thanks to recent improvements in NMR instrumentation, pulse sequence design, and sample preparation, a panoply of new NMR tools has become available for atomic resolution characterization of intrinsically disordered proteins (IDPs) that are optimized for the particular chemical and spectroscopic properties of these molecules. A wide range of NMR observables can now be measured on increasingly complex IDPs that report on their structural and dynamic properties in isolation, as part of a larger complex, or even inside an entire living cell. Herein we present basic NMR concepts, as well as optimised tools available for the study of IDPs in solution. In particular, the following sections are discussed hereafter: a short introduction to NMR spectroscopy and instrumentation (Sect. 3.1), the effect of order and disorder on NMR observables (Sect. 3.2), particular challenges and bottlenecks for NMR studies of IDPs (Sect. 3.3), 2D HN and CON NMR experiments: the fingerprint of an IDP (Sect. 3.4), tools for overcoming major bottlenecks of IDP NMR studies (Sect. 3.5), 13C detected experiments (Sect. 3.6), from 2D to 3D: from simple snapshots to site-resolved characterization of IDPs (Sect. 3.7), sequential NMR assignment: 3D experiments (Sect. 3.8), high-dimensional NMR experiments (nD, with n>3) (Sect. 3.9) and conclusions and perspectives (Sect. 3.10).
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http://dx.doi.org/10.1007/978-3-319-20164-1_3DOI Listing
December 2015

Longitudinal relaxation properties of (1)H(N) and (1)H(α) determined by direct-detected (13)C NMR experiments to study intrinsically disordered proteins (IDPs).

J Magn Reson 2015 May 12;254:19-26. Epub 2015 Feb 12.

CERM and Department of Chemistry Ugo Schiff, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy. Electronic address:

Intrinsically disordered proteins (IDPs) are functional proteins containing large fragments characterized by high local mobility. Bioinformatic studies have suggested that a significant fraction (more than 30%) of eukaryotic proteins has disordered regions of more than 50 amino acids in length. Hence, NMR methods for the characterization of local compactness and solvent accessibility in such highly disordered proteins are of high importance. Among the available approaches, the HET-SOFAST/BEST experiments (Schanda et al., 2006, Rennella et al., 2014) provide semi-quantitative information by monitoring longitudinal (1)H relaxation of amide protons under different initial conditions. However, when approaching physiological sample conditions, the potential of these amide (1)H detected experiments is reduced due to rapid amide proton solvent exchange. (13)C direct detection methods therefore provide a valuable alternative thanks to a higher chemical shift dispersion and their intrinsic insensitivity toward solvent exchange. Here we present two sets of (13)C-detected experiments, which indirectly measure (1)H(N) and (1)H(α) inversion recovery profiles. The experiments consist of an initial spin inversion-recovery block optimized for selective manipulation of different types of proton spins followed by a CON read-out scheme. The proposed experiments were tested on human α-synuclein and ubiquitin, two representative examples of unfolded and folded proteins.
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http://dx.doi.org/10.1016/j.jmr.2015.01.017DOI Listing
May 2015

"CON-CON" assignment strategy for highly flexible intrinsically disordered proteins.

J Biomol NMR 2014 Dec 19;60(4):209-18. Epub 2014 Oct 19.

CERM and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy.

Intrinsically disordered proteins (IDPs) are a class of highly flexible proteins whose characterization by NMR spectroscopy is complicated by severe spectral overlaps. The development of experiments designed to facilitate the sequence-specific assignment procedure is thus very important to improve the tools for the characterization of IDPs and thus to be able to focus on IDPs of increasing size and complexity. Here, we present and describe the implementation of a set of novel ¹H-detected 5D experiments, (HACA)CON(CACO)NCO(CA)HA, BT-(H)NCO(CAN)CONNH and BT-HN(COCAN)CONNH, optimized for the study of highly flexible IDPs that exploit the best resolved correlations, those involving the carbonyl and nitrogen nuclei of neighboring amino acids, to achieve sequence-specific resonance assignment. Together with the analogous recently proposed pulse schemes based on ¹³C detection, they form a complete set of experiments for sequence-specific assignment of highly flexible IDPs. Depending on the particular sample conditions (concentration, lifetime, pH, temperature, etc.), these experiments present certain advantages and disadvantages that will be discussed. Needless to say, that the availability of a variety of complementary experiments will be important for accurate determination of resonance frequencies in complex IDPs.
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http://dx.doi.org/10.1007/s10858-014-9867-6DOI Listing
December 2014

Measuring hydrogen exchange in proteins by selective water saturation in (1)H- (15)N SOFAST/BEST-type experiments: advantages and limitations.

J Biomol NMR 2014 Nov 31;60(2-3):99-107. Epub 2014 Aug 31.

Institut de Biologie Structurale, Université Grenoble 1, 71 avenue des Martyrs, 38044, Grenoble Cedex 9, France.

HET(ex)-SOFAST NMR (Schanda et al. in J Biomol NMR 33:199-211, 2006) has been proposed some years ago as a fast and sensitive method for semi-quantitative measurement of site-specific amide-water hydrogen exchange effects along the backbone of proteins. Here we extend this concept to BEST readout sequences that provide a better resolution at the expense of some loss in sensitivity. We discuss the theoretical background and implementation of the experiment, and demonstrate its performance for an intrinsically disordered protein, 2 well folded globular proteins, and a transiently populated folding intermediate state. We also provide a critical evaluation of the level of accuracy that can be obtained when extracting quantitative exchange rates from HET(ex) NMR measurements.
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http://dx.doi.org/10.1007/s10858-014-9857-8DOI Listing
November 2014

HNCA+, HNCO+, and HNCACB+ experiments: improved performance by simultaneous detection of orthogonal coherence transfer pathways.

J Biomol NMR 2014 Sep 24;60(1):1-9. Epub 2014 Jul 24.

Institut de Biologie Structurale, Université Grenoble 1, 71 Avenue des Martyrs, 38044, Grenoble Cedex 9, France.

Three experiments, BEST-TROSY HNCA+, HNCO+ and HNCACB+ are presented for sequential backbone resonance assignment of (13)C, (15)N labelled proteins. The novelty of these experiments with respect to conventional pulse sequences is the detection of additional orthogonal coherence transfer pathways that results in enhanced sensitivity for sequential correlations without significantly compromising the intensity of intra-residue correlation peaks. In addition, a 2-step phase cycle separates peaks originating from the orthogonal coherence transfer pathways in 2 sub-spectra, thus providing similar information as obtained from performing a pair of sequential and intra-residue correlation experiments.
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http://dx.doi.org/10.1007/s10858-014-9847-xDOI Listing
September 2014

NMR spectroscopic studies of intrinsically disordered proteins at near-physiological conditions.

Angew Chem Int Ed Engl 2013 Nov 20;52(45):11808-12. Epub 2013 Sep 20.

Bruker BioSpin AG, Industriestrasse 26, 8117 Fällanden (Switzerland).

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http://dx.doi.org/10.1002/anie.201304272DOI Listing
November 2013

Interaction of nonstructural protein 5A of the hepatitis C virus with Src homology 3 domains using noncanonical binding sites.

Biochemistry 2013 Sep 26;52(36):6160-8. Epub 2013 Aug 26.

Institut de Biologie Structurale, Université Grenoble 1 , 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France.

Src homology 3 (SH3) domains are widely known for their ability to interact with other proteins using the canonical PxxP binding motif. Besides those well-characterized interaction modes, there is an increasing number of SH3 domain-containing complexes that lack this motif. Here we characterize the interaction of SH3 domains, in particular the Bin1-SH3 domain, with the intrinsically disordered part of nonstructural protein 5A of the hepatitis C virus using noncanonical binding sites in addition to its PxxP motif. These binding regions partially overlap with regions that have previously been identified as having an increased propensity to form α-helices. Remarkably, upon interaction with the Bin1-SH3 domain, the α-helical propensity decreases and a fuzzy complex is formed.
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http://dx.doi.org/10.1021/bi400363vDOI Listing
September 2013

Fast real-time NMR methods for characterizing short-lived molecular states.

Chemphyschem 2013 Sep 15;14(13):3059-70. Epub 2013 Jul 15.

Biomolecular NMR spectroscopy group Institut de Biologie Structurale, Université Grenoble 1, CNRS, CEA, 41, rue Jules Horowitz, 38027 Grenoble Cedex 1 (France), Fax: (+33) 4 38 78 54 94.

The characterization of both the structure and the conformational dynamics of biological macromolecules, namely proteins and nucleic acids, is required for understanding the molecular mechanisms underlying physiological function and disease. Molecular dynamics involves the transient departure from the ground-state structures to populate short-lived excited state conformations that can play important functional roles. Real-time multi-dimensional NMR spectroscopy represents a unique tool for investigating dynamic molecular processes occurring on time scales of seconds or longer, providing atomic-resolution information about short-lived states. In this minireview, we discuss recent progress made in the field of fast real-time multidimensional NMR. The potential of these new methods is illustrated for several biomolecular systems that have recently been studied by fast real-time multidimensional NMR.
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http://dx.doi.org/10.1002/cphc.201300339DOI Listing
September 2013

Highly efficient NMR assignment of intrinsically disordered proteins: application to B- and T cell receptor domains.

PLoS One 2013 7;8(5):e62947. Epub 2013 May 7.

Swedish NMR Centre, University of Gothenburg, Gothenburg, Sweden.

We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062947PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647075PMC
December 2013

BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins.

J Biomol NMR 2013 Apr 24;55(4):311-21. Epub 2013 Feb 24.

Institut de Biologie Structurale, Université Grenoble 1, 41 Rue Jules Horowitz, 38027 Grenoble Cedex 1, France.

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.
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http://dx.doi.org/10.1007/s10858-013-9715-0DOI Listing
April 2013

Real-time NMR characterization of structure and dynamics in a transiently populated protein folding intermediate.

J Am Chem Soc 2012 May 7;134(19):8066-9. Epub 2012 May 7.

Institut de Biologie Structurale, Université Grenoble 1, CEA, CNRS, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France.

Recent advances in NMR spectroscopy and the availability of high magnetic field strengths now offer the possibility to record real-time 3D NMR spectra of short-lived protein states, e.g., states that become transiently populated during protein folding. Here we present a strategy for obtaining sequential NMR assignments as well as atom-resolved information on structural and dynamic features within a folding intermediate of the amyloidogenic protein β2-microglobulin that has a half-lifetime of only 20 min.
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http://dx.doi.org/10.1021/ja302598jDOI Listing
May 2012

Transient structure and SH3 interaction sites in an intrinsically disordered fragment of the hepatitis C virus protein NS5A.

J Mol Biol 2012 Jul 26;420(4-5):310-23. Epub 2012 Apr 26.

Institut de Biologie Structurale, Université Grenoble 1, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France.

Understanding the molecular mechanisms involved in virus replication and particle assembly is of primary fundamental and biomedical importance. Intrinsic conformational disorder plays a prominent role in viral proteins and their interaction with other viral and host cell proteins via transiently populated structural elements. Here, we report on the results of an investigation of an intrinsically disordered 188-residue fragment of the hepatitis C virus non-structural protein 5A (NS5A), which contains a classical poly-proline Src homology 3 (SH3) binding motif, using sensitivity- and resolution-optimized multidimensional NMR methods, complemented by small-angle X-ray scattering data. Our study provides detailed atomic-resolution information on transient local and long-range structure, as well as fast time scale dynamics in this NS5A fragment. In addition, we could characterize two distinct interaction modes with the SH3 domain of Bin1 (bridging integrator protein 1), a pro-apoptotic tumor suppressor. Despite being largely disordered, the protein contains three regions that transiently adopt α-helical structures, partly stabilized by long-range tertiary interactions. Two of these transient α-helices form a noncanonical SH3-binding motif, which allows low-affinity SH3 binding. Our results contribute to a better understanding of the role of the NS5A protein during hepatitis C virus infection. The present work also highlights the power of NMR spectroscopy to characterize multiple binding events including short-lived transient interactions between globular and highly disordered proteins.
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http://dx.doi.org/10.1016/j.jmb.2012.04.023DOI Listing
July 2012

Selective isotopic unlabeling of proteins using metabolic precursors: application to NMR assignment of intrinsically disordered proteins.

Chembiochem 2012 Mar 8;13(5):732-9. Epub 2012 Mar 8.

Instituto de Biología Molecular y Celular de Rosario, Suipacha 531, S2002LRK Rosario, Argentina.

Selective isotopic unlabeling of proteins can provide important residue-type information as well as reduce congestion of NMR spectra. However, metabolic scrambling often complicates the final isotope-labeling pattern. Here, an array of metabolic precursors is used to perform robust, residue-specific unlabeling of proteins. The resulting isotopic-labeling patterns are predictable and nicely complement NMR experiments that differentiate residue types. This approach has widespread applications, but it is particularly relevant for proteins that lack sequence complexity or a defined tertiary structure.
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http://dx.doi.org/10.1002/cbic.201100678DOI Listing
March 2012

Fast protein backbone NMR resonance assignment using the BATCH strategy.

Methods Mol Biol 2012 ;831:407-28

Institut de Biologie Structurale - Jean-Pierre Ebel, CNRS, CEA, UJF, UMR5075, Grenoble Cedex, France.

Probing protein structure, dynamics, and interaction surfaces by NMR requires initial backbone resonance assignment. The protocol for this step has been progressively developed in the last 15 years to provide robust assignments. However, even in the case of favorable conditions (high field magnets and cryogenically cooled probes, small globular proteins, high sample concentration), the assignment step generally takes several days of data collection and analysis, thus precluding studies of unstable proteins and limiting high-throughput applications. Recently, we have introduced the BATCH strategy for fast protein backbone resonance assignment. BATCH benefits from the combination of several tools (BEST/ASCOM/Targeted-Sampling/COBRA/HADAMAC) for time-optimized and highly automated NMR data acquisition, processing, and analysis. In this chapter, we discuss the individual steps of the BATCH method and describe its practical implementation to obtain the backbone resonance assignment of small globular proteins in a few hours of time.
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http://dx.doi.org/10.1007/978-1-61779-480-3_21DOI Listing
April 2012

iHADAMAC: a complementary tool for sequential resonance assignment of globular and highly disordered proteins.

J Magn Reson 2012 Jan 9;214(1):329-34. Epub 2011 Nov 9.

Institut de Biologie Structurale, Université Grenoble 1, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France.

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.
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http://dx.doi.org/10.1016/j.jmr.2011.10.019DOI Listing
January 2012

Rapid measurement of residual dipolar couplings for fast fold elucidation of proteins.

J Biomol NMR 2011 Nov 14;51(3):369-78. Epub 2011 Sep 14.

Institut de Biologie Structurale, Jean-Pierre Ebel CNRS/CEA/UJF, 41 rue Jules Horowitz, 38027 Grenoble Cedex, France.

It has been demonstrated that protein folds can be determined using appropriate computational protocols with NMR chemical shifts as the sole source of experimental restraints. While such approaches are very promising they still suffer from low convergence resulting in long computation times to achieve accurate results. Here we present a suite of time- and sensitivity optimized NMR experiments for rapid measurement of up to six RDCs per residue. Including such an RDC data set, measured in less than 24 h on a single aligned protein sample, greatly improves convergence of the Rosetta-NMR protocol, allowing for overnight fold calculation of small proteins. We demonstrate the performance of our fast fold calculation approach for ubiquitin as a test case, and for two RNA-binding domains of the plant protein HYL1. Structure calculations based on simulated RDC data highlight the importance of an accurate and precise set of several complementary RDCs as additional input restraints for high-quality de novo structure determination.
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http://dx.doi.org/10.1007/s10858-011-9567-4DOI Listing
November 2011
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