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  • 1.
    Ahlner, Alexandra
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Andresen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Khan, Shahid N.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Kay, Lewis E.
    Departments of Medical Genetics, Biochemistry and Chemistry, The University of Toronto, Canada.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Fractional enrichment of proteins using [2-13C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity2015In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 62, no 3, p. 341-351Article in journal (Refereed)
    Abstract [en]

    A selective isotope labeling scheme based on the utilization of [2-13C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13Cα chemical shifts using Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-13C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state 13Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s−1, despite the small fraction of 13Cα–13Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using 13Cα spin probes.

  • 2.
    Ahlner, Alexandra
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Carlsson, Mats
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    PINT: a software for integration of peak volumes and extraction of relaxation rates2013In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 56, no 3, p. 191-202Article in journal (Refereed)
    Abstract [en]

    We present the software Peak INTegration (PINT), designed to perform integration of peaks in NMR spectra. The program is very simple to run, yet powerful enough to handle complicated spectra. Peaks are integrated by fitting predefined line shapes to experimental data and the fitting can be customized to deal with, for instance, heavily overlapped peaks. The results can be inspected visually, which facilitates systematic optimization of the line shape fitting. Finally, integrated peak volumes can be used to extract parameters such as relaxation rates and information about low populated states. The utility of PINT is demonstrated by applications to the 59 residue SH3 domain of the yeast protein Abp1p and the 289 residue kinase domain of murine EphB2.

  • 3.
    Lundström, Patrik
    et al.
    Lund University, Department of Biophysical Chemistry.
    Akke, Mikael
    Lund University, Department of Biophysical Chemistry.
    Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins2005In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 32, no 2, p. 163-173Article in journal (Refereed)
    Abstract [en]

    NMR spin relaxation in the rotating frame (R-1 rho) is a unique method for atomic-resolution characterization of conformational ( chemical) exchange processes occurring on the microsecond time scale. Here, we use amide H-1 off-resonance R-1 rho relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using N-15 or C-13 relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The H-1 off-resonance R-1 rho data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 mu s. This is in contrast to the case for N-15, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly N-15-enriched samples that were either highly enriched in H-2 or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the H-1 transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present H-1 off-resonance R-1 rho experiments are in excellent agreement with those determined previously using a combination of N-15 laboratory-frame and off-resonance R-1 rho relaxation data, with average values of <tau(ex)> = 19 +/- 7 and 21 +/- 3 mu s, respectively.

  • 4.
    Lundström, Patrik
    et al.
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Hansen, D. Flemming
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Kay, Lewis E.
    University of Toronto, Departments of Biochemistry, Chemistry and Medical Genetics.
    Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively C-13 labeled samples2008In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 42, no 1, p. 35-47Article in journal (Refereed)
    Abstract [en]

    Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated C-13 spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (CO)-C-13 experiments however the large separation between (CO)-C-13 and C-13(alpha) chemical shifts offers hope that robust (CO)-C-13 dispersion profiles can be recorded on uniformly C-13 labeled samples, leading to the extraction of accurate (CO)-C-13 chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-C-13]-pyruvate and (NaHCO3,)-C-13 or uniformly labeled, generated from C-13-glucose. Very similar (CO)-C-13 chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.

  • 5.
    Lundström, Patrik
    et al.
    University of Toronto, ON, Canada.
    Teilum, Kaare
    Lund University, Sweden .
    Carstensen, Tommy
    Lund University, Sweden .
    Bezsonova, Irina
    University of Toronto, ON, Canada.
    Wiesner, Silke
    University of Toronto, ON, Canada.
    Hansen, D. Flemming
    University of Toronto, ON, Canada.
    Religa, Tomasz L.
    MRC, University of Cambridge, UK.
    Akke, Mikael
    Lund University, Sweden .
    Kay, Lewis E.
    University of Toronto, ON, Canada.
    Fractional C-13 enrichment of isolated carbons using [1-C-13]- or [2-C-13]-glucose facilitates the accurate measurement of dynamics at backbone C-alpha and side-chain methyl positions in proteins2007In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 38, no 3, p. 199-212Article in journal (Refereed)
    Abstract [en]

    A simple labeling approach is presented based on protein expression in [1-C-13]- or [2-C-13]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C-alpha sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta 1) are produced with isolated C-13 spins (i.e., no C-13-C-13 one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-alpha sites are labeled without concomitant labeling at C-beta positions for 17 of the common 20 amino acids and there are no cases for which C-13(alpha)-(CO)-C-13 spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone N-15 studies. The utility of the labeling is established by recording C-13 R-1 rho and CPMG-based experiments on a number of different protein systems.

  • 6.
    Lundström, Patrik
    et al.
    University of Toronto, ON, Canada.
    Vallurupalli, Pramodh
    University of Toronto, ON, Canada.
    Religa, Tomasz
    MRC, University of Cambridge, UK.
    Dahlquist, Fredrick W.
    University of California at Santa Barbara, USA.
    Kay, Lewis E.
    University of Toronto, ON, Canada.
    A single-quantum methyl C-13-relaxation dispersion experiment with improved sensitivity2007In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 38, no 1, p. 79-88Article in journal (Refereed)
    Abstract [en]

    A pulse sequence is described for recording single-quantum (13)C-methyl relaxation dispersion profiles of (13)C-selectively labeled methyl groups in proteins that offers significant improvements in sensitivity relative to existing approaches where initial magnetization derives from (13)C polarization. Sensitivity gains in the new experiment are achieved by making use of polarization from (1)H spins and (1)H --> (13)C --> (1)H type magnetization transfers. Its utility has been established by applications involving three different protein systems ranging in molecular weight from 8 to 28 kDa, produced using a number of different selective labeling approaches. In all cases exchange parameters from both (13)C-->(1)H and (1)H --> (13)C --> (1)H classes of experiment are in good agreement, with gains in sensitivity of between 1.7 and 4-fold realized using the new scheme.

  • 7.
    Mayzel, Maxim
    et al.
    University of Gothenburg, Sweden.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Orekhov, Vladislav Y.
    University of Gothenburg, Sweden.
    Measurement of protein backbone (CO)-C-13 and N-15 relaxation dispersion at high resolution2017In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 69, no 1Article in journal (Refereed)
    Abstract [en]

    Peak overlap in crowded regions of two-dimensional spectra prevents characterization of dynamics for many sites of interest in globular and intrinsically disordered proteins. We present new three-dimensional pulse sequences for measurement of Carr-Purcell-Meiboom-Gill relaxation dispersions at backbone nitrogen and carbonyl positions. To alleviate increase in the measurement time associated with the additional spectral dimension, we use non-uniform sampling in combination with two distinct methods of spectrum reconstruction: compressed sensing and co-processing with multi-dimensional decomposition. The new methodology was validated using disordered protein CD79A from B-cell receptor and an SH3 domain from Abp1p in exchange between its free form and bound to a peptide from the protein Ark1p. We show that, while providing much better resolution, the 3D NUS experiments give the similar accuracy and precision of the dynamic parameters to ones obtained using traditional 2D experiments. Furthermore, we show that jackknife resampling of the spectra yields robust estimates of peak intensities errors, eliminating the need for recording duplicate data points.

  • 8.
    Niklasson, Markus
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Otten, Renee
    Howard Hughes Medical Institute and Department of Biochemistry, Brandeis University, Waltham, MA, USA..
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Andrésen, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Schlagnitweit, Judith
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    Petzold, Katja
    Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Comprehensive analysis of NMR data using advanced line shape fitting.2017In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 69, no 2, p. 93-99Article in journal (Refereed)
    Abstract [en]

    NMR spectroscopy is uniquely suited for atomic resolution studies of biomolecules such as proteins, nucleic acids and metabolites, since detailed information on structure and dynamics are encoded in positions and line shapes of peaks in NMR spectra. Unfortunately, accurate determination of these parameters is often complicated and time consuming, in part due to the need for different software at the various analysis steps and for validating the results. Here, we present an integrated, cross-platform and open-source software that is significantly more versatile than the typical line shape fitting application. The software is a completely redesigned version of PINT ( https://pint-nmr.github.io/PINT/ ). It features a graphical user interface and includes functionality for peak picking, editing of peak lists and line shape fitting. In addition, the obtained peak intensities can be used directly to extract, for instance, relaxation rates, heteronuclear NOE values and exchange parameters. In contrast to most available software the entire process from spectral visualization to preparation of publication-ready figures is done solely using PINT and often within minutes, thereby, increasing productivity for users of all experience levels. Unique to the software are also the outstanding tools for evaluating the quality of the fitting results and extensive, but easy-to-use, customization of the fitting protocol and graphical output. In this communication, we describe the features of the new version of PINT and benchmark its performance.

  • 9.
    Vallurupalli, Pramodh
    et al.
    University of Toronto, ON, Canada.
    Hansen, D. Flemming
    University of Toronto, ON, Canada.
    Lundström, Patrik
    University of Toronto, ON, Canada.
    Kay, Lewis E.
    University of Toronto, ON, Canada.
    CPMG relaxation dispersion NMR experiments measuring glycine H-1(alpha) and C-13(alpha) chemical shifts in the 'invisible' excited states of proteins2009In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 45, no 1-2, p. 45-55Article in journal (Refereed)
    Abstract [en]

    Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments are extremely powerful for characterizing millisecond time-scale conformational exchange processes in biomolecules. A large number of such CPMG experiments have now emerged for measuring protein backbone chemical shifts of sparsely populated (> 0.5%), excited state conformers that cannot be directly detected in NMR spectra and that are invisible to most other biophysical methods as well. A notable deficiency is, however, the absence of CPMG experiments for measurement of H-1(alpha) and C-13(alpha) chemical shifts of glycine residues in the excited state that reflects the fact that in this case the H-1(alpha), C-13(alpha) spins form a three-spin system that is more complex than the AX H-1(alpha)-C-13(alpha) spin systems in the other amino acids. Here pulse sequences for recording H-1(alpha) and C-13(alpha) CPMG relaxation dispersion profiles derived from glycine residues are presented that provide information from which H-1(alpha), C-13(alpha) chemical shifts can be obtained. The utility of these experiments is demonstrated by an application to a mutant of T4 lysozyme that undergoes a millisecond time-scale exchange process facilitating the binding of hydrophobic ligands to an internal cavity in the protein.

  • 10.
    Weininger, Ulrich
    et al.
    Lund University, Sweden .
    Blissing, Annica T.
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Hennig, Janosch
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
    Ahlner, Alexandra
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Liu, Zhihong
    University of Calgary, Canada .
    Vogel, Hans J.
    University of Calgary, Canada .
    Akke, Mikael
    Lund University, Sweden .
    Lundström, Patrik
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
    Protein conformational exchange measured by H-1 R-1 rho relaxation dispersion of methyl groups2013In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 57, no 1, p. 47-55Article in journal (Refereed)
    Abstract [en]

    Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for H-1 spins in methyl (CHD2)-C-13 groups, which improves the characterization of fast exchange processes. The influence of H-1-H-1 rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R (1 rho) relaxation data acquired with tilt angles of 90A degrees and 35A degrees, in which the ROE is maximal and minimal, respectively, and on samples containing different H-1 densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any H-1 relaxation dispersion of its methyl groups at 10 or 25 A degrees C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by H-1 and C-13 CPMG experiments. The present R (1 rho) experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.

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