Simultaneous measurement of residual dipolar couplings for proteins in complex using the isotopically discriminated NMR approach

Bermel, Wolfgang; Tkach, Elena N.; Sobol, A. A.; Golovanov, Alexander P.

doi:10.1021/ja901602c

Cited by 9 publications

(11 citation statements)

References 36 publications

(60 reference statements)

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“…Conceptually, it is also closely related to isotopically discriminated (IDIS) NMR [59, 91] as well as carbonyl carbon label selective (CCLS) [58] and especially dual carbon label selective (DCLS) [60, 92] schemes. In the former, 2D HN(CO) based pulse sequences with and without evolution of 1 J ( 15 N, 13 C’) for a period equaling the inverse coupling constant are applied to mixtures of uniformly 15 N- and 13 C/ 15 N-labeled proteins.…”

Section: Resultsmentioning

confidence: 99%

Time-shared experiments for efficient assignment of triple-selectively labeled proteins

Löhr

Laguerre

Bock

et al. 2014

Journal of Magnetic Resonance

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Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of 13C 90° pulses allows to either select or discriminate against 13C’ or 13Cα spins coupled to 15N. As a result, 1H-15N correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns.

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Section: Resultsmentioning

confidence: 99%

Time-shared experiments for efficient assignment of triple-selectively labeled proteins

Löhr

Laguerre

Bock

et al. 2014

Journal of Magnetic Resonance

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“…Although both proteins are then 15 N-labelled and will yield (overlapping) 2D 1 H, 15 N correlation spectra in a single experiment, the presence of J-couplings to 13 C atoms allows the capability to edit the spectrum for or against the double labelled component, thereby permitting separation of the two component subspectra -the pulse sequences that have been developed for this purpose have been dubbed 'isotope-discriminated NMR' or IDIS-NMR. Under these conditions the spectroscopist can be sure to measure certain NMR parameters (chemical shifts, 15 N-relaxation times, residual dipolar couplings) for the two components of a complex under intrinsically 'matched' conditions, albeit perhaps with a small loss of sensitivity due to the additional RF pulses required [30][31][32].…”

Section: Spectral Simplification Through Differential Isotope Labellingmentioning

confidence: 99%

“…Alternatively, the experimenter can appeal to the concept of combined single-/double-isotope labelling described above [30] combined with IDIS-type NMR pulse sequences for the extraction of the RDC values. In this way the RDC values obtained for each component of the complex are obtained under equivalent alignment conditions for each step in the titration [31], effectively eliminating the potential for mismatch in buffer or alignment conditions that will almost inevitably occur for measurements made of separately labelled samples.…”

Section: Residual Dipolar Couplingsmentioning

confidence: 99%

Macromolecular Complexes

Driscoll¹

2011

Protein NMR Spectroscopy: Practical Techniques and Applications

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It is clear that applications of solution state NMR spectroscopy to the interaction of small molecule ligands with biological macromolecules has proved immensely useful in a number of fields of activity, ranging from fundamental biological research to the commercial development of therapeutic agents. In a similar vein it is undeniably the case that NMR investigations of the mutual interactions between macromolecules themselves have provided insights that, oftentimes, are not obtainable by other methods or complement those garnered by alternative approaches. There is significant overlap of many of the basic principles of the application of NMR to complexes of macromolecules with small molecules, and macromolecular assemblies, though clearly a major factor in the latter case is that the approach is usually constrained by the tendency of the target to yield broader resonances as a result of slow(er) overall tumbling, reflecting the fundamental hydrodynamic characteristics of globular assemblies. Combined with the higher intrinsic overall complexity of the spectra of such systems (because of the potential number of distinct nuclear spins), considerations of how to handle such line-broadening tend to dominate the experimental design and one typically appeals to multiple paradigms to lay siege to this type of problem.It is transparent to anyone with exposure to solution state applications of NMR spectroscopy that this is an area that is always going to receive tough competition from our colleagues who approach such targets by X-ray crystallography, not least because the latter technique does not suffer (to first order) from the impact of increased molecular size. Nevertheless NMR provides a purely solution state method that can compete with, Protein NMR Spectroscopy: Practical Techniques and Applications, First Edition. Edited

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“…When a binary mixture of proteins is studied, the signal-to-noise (S/N) resulting from the pulse sequence (Figure 1A) used to detect the 15 N-labeled species is √2 higher than approaches that rely on a HNCO-based sequence,18 as has been proposed recently 20,21. In addition, the time period in which magnetization is transverse in HNCO-based experiments is ∼68 ms, whereas the sequences presented here only require half this time period (∼33 ms) for a binary mixture or ∼49 ms for a ternary mixture.…”

mentioning

confidence: 90%

“…Unlike recently reported schemes,20,21 the pulse sequence used in this approach is shorter, and therefore provides higher sensitivity, particularly for large systems. Additionally, the approach supports the unambiguous identification of spectral information from each subunit of a binary or ternary complex.…”

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confidence: 99%

One-Sample Approach to Determine the Relative Orientations of Proteins in Ternary and Binary Complexes from Residual Dipolar Coupling Measurements

et al. 2009

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We present a procedure that supports the acquisition of 1 H-15 N residual dipolar coupling (RDC) values for individual subunits in binary or ternary protein assemblies from a single experimental sample. Our method relies on asymmetric labeling of each subunit with the following scheme: species A uniformly with 15 N, species B uniformly with 15 N and 13 C, and species C uniformly with 15 N but selectively with 13 C′ or 13 C α . Because only a single sample is required, the approach obviates the need for preparing multiple samples and eliminates potential errors introduced from differences in sample conditions. Because numerous biological processes rely on protein assemblies or transient interactions, this method should be well suited for a wide range of future applications.NMR spectroscopy is a powerful method for analyzing changes at the atomic level in the structure and dynamics of macromolecular complexes. 1-4 NMR distance restraints used to define the conformational space of protein-protein assemblies are commonly derived from intermolecular nuclear Overhauser effects, 5-7 paramagnetic relaxation enhancements, 8,9 cross-saturation, 10 and chemical shift perturbations. 11 However, because full analysis requires spectral information from each partner in the complex, multiple samples are used for such measurements. Thus the full atomic details of protein assemblies, which only NMR can offer in solution, can be time and cost restrictive. Other strategies have exploited amino acid selective 12 or asymmetric labeling patterns 13,14 to facilitate measurements of intermolecular distances. Unfortunately, only the interface is characterized and the full backbone conformation and relative orientation of protein-protein complexes is not defined. In some cases, the approach requires sequences which are insensitive for large assemblies with inherently short transverse relaxation times (T 2 ).The conformational space of macromolecular complexes also can be determined from orientational restraints derived from residual dipolar couplings (RDCs) under weak The implementation of a spin-echo difference during a constant time period of an HSQC was originally introduced by Bax and co-workers 17 for the measurement of sidechain dihedral angles. We recently applied a similar constant time spin-echo filter element to replace a two-dimensional version of the triple resonance HNCO pulse sequence to identify sequential pairs of amino acids in large proteins and enzymes, 18 and extended this idea to enable chemical shift perturbation mapping of samples containing three isotopically labeled species in solution. 19 In this Communication we report an approach that, in combination with an asymmetric isotopic labeling scheme, enables the simultaneous measurements of RDCs from subunits of binary and ternary complexes with high sensitivity. Unlike recently reported schemes, 20,21 the pulse sequence used in this approach is shorter, and therefore provides higher sensitivity, particularly for large systems. Additionally, the approach supp...

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Simultaneous measurement of residual dipolar couplings for proteins in complex using the isotopically discriminated NMR approach

Cited by 9 publications

References 36 publications

Time-shared experiments for efficient assignment of triple-selectively labeled proteins

Time-shared experiments for efficient assignment of triple-selectively labeled proteins

Macromolecular Complexes

One-Sample Approach to Determine the Relative Orientations of Proteins in Ternary and Binary Complexes from Residual Dipolar Coupling Measurements

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