Longitudinal <sup>1</sup>H Relaxation Optimization in TROSY NMR Spectroscopy

Pervushin, Konstantin; Vögeli, Beat; Eletsky, Alexander

doi:10.1021/ja027149q

Cited by 170 publications

(142 citation statements)

References 30 publications

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“…This general signal loss can be minimized by maintaining a high steady-state water polarization throughout the experiment, which is usually achieved using selective ''flip-back'' pulses on the water resonance . Similar effects have been observed between the aliphatic proton resonances and the remainder of the spectrum in fully protonated large molecular systems (Akasaka et al, 1978), and it has been demonstrated that pulse sequences avoiding saturation of the covalently bound aliphatic protons yield more signal (Pervushin et al, 2002).…”

Section: Introductionsupporting

confidence: 63%

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

et al. 2005

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In large molecular structures, the magnetization of all hydrogen atoms in the solute is strongly coupled to the water magnetization through chemical exchange between solvent water and labile protons of macromolecular components, and through dipole-dipole interactions and the associated ''spin diffusion'' due to slow molecular tumbling. In NMR experiments with such systems, the extent of the water polarization is thus of utmost importance. This paper presents a formalism that describes the propagation of the water polarization during the course of different NMR experiments, and then compares the results of model calculations for optimized water polarization with experimental data. It thus demonstrates that NMR spectra of large molecular structures can be improved with the use of paramagnetic spin relaxation agents which selectively enhance the relaxation of water protons, so that a substantial gain in signal-to-noise can be achieved. The presently proposed use of a relaxation agent can also replace the water flip-back pulses when working with structures larger than about 30 kDa. This may be a valid alternative in situations where flip-back pulses are difficult to introduce into the overall experimental scheme, or where they would interfere with other requirements of the NMR experiment.

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Section: Introductionsupporting

confidence: 63%

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

et al. 2005

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“…First, the number of scans required to sample the multidimensional time space may be reduced by using spectral aliasing, nonlinear data sampling techniques, spatial frequency encoding, or Hadamard-type frequency-space spectroscopy (7,8). Second, accelerating the recovery of spin polarization between scans by means of optimized pulse sequences (9)(10)(11) or the addition of relaxation agents to the solution (12, 13) allows higher repetition rates of the pulse sequence. Here, we propose a fluid turbulence-adapted (FTA) version of 1 H-15 N 2D SOFAST-heteronuclear multiple quantum coherence (HMQC) (10,14) to follow a kinetic process for individual amide sites in a protein [see supporting information (SI) Fig.…”

Section: Resultsmentioning

confidence: 99%

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Schanda

Forge

Brutscher

2007

Proc. Natl. Acad. Sci. U.S.A.

152

151

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Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of ␣-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of ␣-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes.hydrogen/deuterium exchange ͉ molecular kinetics ͉ ␣-lactalbumin ͉ ubiquitin A detailed description of the structural changes occurring during the folding and unfolding of proteins still remains a challenging objective in biophysics. The understanding of the fundamental mechanisms of the folding process will also shed light on the factors leading to protein misfolding responsible for neurodegenerative diseases such as Alzheimer's and Parkinson's diseases (1). The ideal method to study protein folding/unfolding provides structural information at atomic resolution and is sensitive to changes occurring on time scales ranging from microseconds to minutes, a task that no single technique is able to fulfill. NMR spectroscopy is especially well adapted to obtain detailed atomistic information about the mechanisms, kinetics, and energetics of the folding/unfolding process for virtually every nuclear site in the protein. NMR methods are sensitive to molecular dynamics occurring over a wide range of time scales (Fig. 1a). Although steady-state NMR methods are well suited to characterize equilibrium molecular dynamics occurring on a subsecond time scale (2, 3), unidirectional processes are best studied by real-time NMR (4, 5), where a series of NMR spectra is recorded during the reaction (Fig. 1b). Two difficulties have so far hampered the widespread application of real-time NMR to the study of protein folding. The first problem is the low intrinsic sensitivity of NMR at ambient temperature, a consequence of the small transition energies ...

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“…As previously exploited in LTROSY (Pervushin et al, 2002) and SOFAST-HMQC (Schanda and Brutscher, 2005), the substantial H u polarisation recovered by the presented efb schemes can be used straightaway to indirectly replenish depleted H N polarisation during the subsequent interscan delay s (defined as the total delay between last and first proton pulse in consecutive scans). This constructive effect of dipolar H u !…”

Section: Accelerated H N Relaxation and Sensitivity Enhancementmentioning

confidence: 93%

“…The spin temperature of the ambient proton lattice thus importantly affects re-equilibration of the signal-generating proton subset, where T 1 relaxation progresses substantially faster under selective (with all except the selected proton spins near thermal equilibrium) than under unselective conditions (with uniformly depleted initial magnetisation for all protons). Experimentally cooling the spin temperature of the proton lattice can thus afford sizeable sensitivity gains through crossrelaxation accelerated polarisation recovery, as recently demonstrated for TROSY-type experiments (Pervushin et al, 2002) and 2D HMQC (Schanda and Brutscher, 2005).…”

Section: Introductionmentioning

confidence: 88%

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Extended Flip-back Schemes for Sensitivity Enhancement in Multidimensional HSQC-type Out-and-back Experiments

2005

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In many NMR experiments, only polarisation of a limited sub-set of all protons is converted into observable coherence. As recently shown by the ''longitudinal'' TROSY implementation (Pervushin et al. (2002) J. Am. Chem. Soc., 124, 12898-12902) and SOFAST-HMQC (Schanda and Brutscher (2005) J. Am. Chem. Soc., 127, 8014-8015), recovery of unused polarisation can be used indirectly and unspecifically to cool the proton lattice and, thus, accelerate re-equilibration for the selected proton subset. Here we illustrate transfer of this principle to HSQC-based multi-dimensional out-and-back experiments that exploit only polarisation of 15 N-bound protons. The presented modifications to the pulse sequences can be implemented broadly and easily, extending standard flip-back of water polarisation to a much larger pool of protons that may comprise all non) 15 N-bound protons. The underlying orthogonal separation of H N polarisation (selected by the main transfer path) from unused H u polarisation (flipped-back on the recovery path) is thereby achieved through positive or negative selection by J-coupling, or using band-selective pulses. In practice, H u polarisation recovery degrades mostly through cumulative pulse imperfections and transverse relaxation; we present, however, strategies to substantially minimise such losses particularly during interim proton decoupling. Depending on the protein's relaxation properties and the extended flipback scheme employed, we recovered up to 60% H u equilibrium polarisation. The concomitant cooling of the proton lattice afforded substantial gains of more than 40%, relative to the water-only flip-back version, in the fast pulsing regime with re-equilibration delays s much shorter than optimal ðs opt ¼ 1:25 Á T 1 ðH N ÞÞ. These would be typically employed if resolution requirements dominate the total measurement time. Contrarily, if sensitivity is limiting and optimal interscan delays s opt can be set (optimal pulsing regime), the best of the presented flip-back schemes may still afford up to ca. 10% absolute sensitivity enhancement.Abbreviations: bsfb -band-selective flip-back; cpd -continuous pulsing decoupling; efb -extended flip-back; H u -unselected proton magnetisation; H C fb -H C flip-back; ufb -universal flip-back; wfb -water flip-back

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Longitudinal ¹H Relaxation Optimization in TROSY NMR Spectroscopy

Cited by 170 publications

References 30 publications

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Extended Flip-back Schemes for Sensitivity Enhancement in Multidimensional HSQC-type Out-and-back Experiments

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Longitudinal 1H Relaxation Optimization in TROSY NMR Spectroscopy

Cited by 170 publications

References 30 publications

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

Extended Flip-back Schemes for Sensitivity Enhancement in Multidimensional HSQC-type Out-and-back Experiments

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Longitudinal ¹H Relaxation Optimization in TROSY NMR Spectroscopy