Novel 13C direct detection experiments, including extension to the third dimension, to perform the complete assignment of proteins

Bermel, Wolfgang; Bertini, Ivano; Felli, Isabella C.; Kümmerle, Rainer; Pierattelli, Roberta

doi:10.1016/j.jmr.2005.08.011

Cited by 116 publications

(88 citation statements)

References 48 publications

(76 reference statements)

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“…[10,56] Even though both of these experiments can in principle be modified for determination of the various NMR observables, we chose 2D CON, since it is well known that 15 N chemical shifts are characterized by a large chemical-shift dispersion. [57,58] To enhance the sensitivity of the experiments without actively labelling proton chemical shifts in any of the dimensions of the NMR experiment, 1 H was used as a starting polarization source ( 1 H start).…”

Section: Resultsmentioning

confidence: 99%

“…[68] Three 2D C'ÀN correlation measurements were performed with the same experimental parameters but different types of experiment ( 13 C start or 1 H start), as well as relaxation delay and number of scans, in order to select the most appropriate experimental approach. In particular, the 13 C-start experiment [56] was acquired with a relaxation delay of 2.5 s and eight scans, and two 1 H start experiments ( Figure 2) were acquired, one with the same parameters and another with a shorter relaxation delay (1.5 s) and a proportionally larger number of scans. The common parameters were: spectral widths of 50 50 ppm for C' and N, respectively, with 1024 512 data points in the direct and indirect acquisition dimensions, including the increments necessary for spin-state selection.…”

Section: Methodsmentioning

confidence: 99%

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Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

et al. 2010

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Provided that (13)C-detected NMR experiments are either preferable or complementary to (1)H detection, we report here tools to determine C(alpha)-C', C'-N, and C(alpha)-H(alpha) residual dipolar couplings on the basis of the CON experiment. The coupling constants determined on ubiquitin are consistent with the subset measured with the (1)H-detected HNCO sequences. Since the utilization of residual dipolar couplings may depend on the mobility of the involved nuclei, we also provide tools to measure longitudinal and transverse relaxation rates of N and C'. This new set of experiments is a further development of a whole strategy based on (13)C direct-detection NMR spectroscopy for the study of biological macromolecules.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

et al. 2010

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“…Many solutions to the problem of homonuclear decoupling were proposed in the literature for different kinds of applications, including band-selective homodecoupling, [70] spin-state selection methods, [71][72][73][74][75][76] and post-acquisition processing protocols. [77] When tested for 13 C direct detection in solution, [78][79][80] as in solid-state applications, [75,76] the approaches that gave the best results were the spin-state selective methods, such as IPAP or S 3 E. These methods rely on the acquisition of different linear combinations of in-phase and anti-phase components, which allows to separate the different multiplet components, to shift them to the center of the original multiplet, and to add them to achieve effective homonuclear decoupling. Several variants were adapted to C' and C a direct detection, allowing simplification of the spectra by removing the large one-bond homonuclear splittings.…”

Section: The Motivationmentioning

confidence: 99%

“…Several variants were adapted to C' and C a direct detection, allowing simplification of the spectra by removing the large one-bond homonuclear splittings. [11,[79][80][81] User-friendly protocols are available to process the data.…”

Section: The Motivationmentioning

confidence: 99%

Recent Advances in Solution NMR: Fast Methods and Heteronuclear Direct Detection

Felli

Brutscher

2009

ChemPhysChem

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Today, NMR spectroscopy is the technique of choice to investigate molecular structure, dynamics, and interactions in solution at atomic resolution. A major limitation of NMR spectroscopy for the study of biological macromolecules such as proteins, nucleic acids, and their complexes, has always been its low sensitivity, a consequence of the weak magnetic spin interactions. Therefore many efforts have been invested in the last decade to improve NMR instrumentation in terms of experimental sensitivity. As a result of these efforts, the availability of high-field magnets, cryogenically cooled probes, and probably in the near future hyperpolarization techniques, the intrinsic NMR sensitivity has increased by at least one order of magnitude. Stimulated by new challenges in the life sciences, these technical improvements have triggered the development of new NMR methods for the study of molecular systems of increasing size and complexity. Herein, we focus on two examples of recently developed NMR methodologies. First, advanced multidimensional data acquisition schemes provide a speed increase of several orders of magnitude. Second, NMR methods based on the direct detection of low-gamma nuclei present a new spectroscopic tool, highly complementary to conventional NMR techniques. These new methods provide powerful new NMR tools for the study of short-lived molecules, large and intrinsically unstructured proteins, paramagnetic systems, as well as for the characterization of molecular kinetic processes at atomic resolution. These examples illustrate how NMR is continuously adapting to the new challenges in the life sciences, with the focus shifting from the characterization of single biomolecules to an integrated view of interacting molecular networks observed at varying levels of biological organization.

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“…Structural studies of biomolecules by NMR have made tremendous progress mainly due to improved recombinant protein expression and 13 C, 15 of novel NMR experiments. 1,2 It has also expanded the scope of NMR with biological macromolecules such as larger proteins by new labeling technology. Whereas NMR structure determination of globular domains of below 20 kDa has increasingly become a routine procedure, resonance assignment of larger proteins can be very time-consuming.…”

Section: Introductionmentioning

confidence: 99%

Use of protein trans‐splicing to produce active and segmentally ²H, ¹⁵N labeled mannuronan C5‐epimerase AlgE4

et al. 2010

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Alginate epimerases are large multidomain proteins capable of epimerising C5 on b-Dmannuronic acid (M) turning it into a-L-guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers. Attempts to study these enzymes with solution-state NMR are hampered by their size-the smallest epimerase, AlgE4, consisting of one A-and one Rmodule, is 58 kDa, resulting in heavy signal overlap impairing the interpretation of NMR spectra. N]-A-R) by protein trans-splicing using the naturally split intein of Nostoc punctiforme. The NMR spectra of native AlgE4 and the ligated versions coincide well proving the conservation of protein structure. The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild-type AlgE4.

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Novel 13C direct detection experiments, including extension to the third dimension, to perform the complete assignment of proteins

Cited by 116 publications

References 48 publications

Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

Recent Advances in Solution NMR: Fast Methods and Heteronuclear Direct Detection

Use of protein trans‐splicing to produce active and segmentally ²H, ¹⁵N labeled mannuronan C5‐epimerase AlgE4

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Novel 13C direct detection experiments, including extension to the third dimension, to perform the complete assignment of proteins

Cited by 116 publications

References 48 publications

Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

Exclusively Heteronuclear NMR Experiments to Obtain Structural and Dynamic Information on Proteins

Recent Advances in Solution NMR: Fast Methods and Heteronuclear Direct Detection

Use of protein trans‐splicing to produce active and segmentally 2H, 15N labeled mannuronan C5‐epimerase AlgE4

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Use of protein trans‐splicing to produce active and segmentally ²H, ¹⁵N labeled mannuronan C5‐epimerase AlgE4