NMR Relaxation Mechanisms for Backbone Carbonyl Carbons in a13C,15N-Labeled Protein

Allard, Peter; Härd, Torleif

doi:10.1006/jmre.1997.1131

Cited by 29 publications

(30 citation statements)

References 30 publications

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“…Less important contributions stem from relaxation induced by DD interactions with nearby protons, whereas cross-relaxation to carbon and nitrogen is insignificant. 11,12,14 Slow modulation of the chemical shift (i.e. chemical or conformational exchange) may contribute a considerable source of transverse relaxation.…”

Section: Relaxation In the Absence Of Rf Irradiationmentioning

confidence: 99%

“…It is interesting in this respect that four out of eight residues that showed significant Figure 5a and b shows part of the 13 C˛-1 H˛region of the CT HSQC spectrum of calbindin D 9k , measured without and with CW irradiation in the C˛region (56 ppm). Residual, J R , and reference splittings, J 0 , were extracted for well-resolved doublets in the two respective 2D spectra, and fitted to Eqn (11). The RF field strength calibration data are shown in Fig.…”

Section: Asp/asnmentioning

confidence: 99%

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Carbonyl ¹³C transverse relaxation measurements to sample protein backbone dynamics

Mulder

Akke

2003

Magnetic Reson in Chemistry

105

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Carbonyl13 C relaxation experiments to study protein backbone dynamics have recently been developed. However, the effect of three-bond 13 C -13 C couplings on transverse relaxation measurements appears not to have been considered, and the potential to detect and quantify motions on the millisecond to microsecond time scale has not been fully explored. The present paper addresses these two issues. Simulations and experiments show that scalar couplings between adjacent backbone carbonyl carbon nuclei and between backbone and side-chain carbonyl/carboxyl carbon atoms in Asp and Asn residues interfere with the accurate determination of transverse relaxation rates by Carr-Purcell-Meiboom-Gill or on-resonance spin-lock measurements. The use of off-resonance radio-frequency fields avoids efficient cross-polarization, and offers a route towards accurate R 1r measurements. In addition, this approach yields dispersion in the transverse relaxation rate as a function of the effective field when conformational exchange is present. In the case of calcium-bound calbindin D 9k , 13 C off-resonance R 1r measurements yielded uniform values of R 2 along the polypeptide chain, indicating homogeneous chemical shift anisotropies and restricted dynamics on the picosecond to nanosecond time scale. Variation of R 2 as a function of the effective spin-lock field strength was not observed for any residue, indicating the absence of large-scale conformational changes of the protein backbone in the millisecond to microsecond time window. The absence of relaxation induced by internal motions on these wide-ranging time scales reinforces the view that calcium-loaded calbindin D 9k is extremely rigid. In contrast, for the C-terminal tryptic fragment of calmodulin containing the E140Q mutation we observed widespread exchange broadening. From the carbonyl transverse relaxation dispersion profile of Asp129 the exchange rate was determined to be 28 000 s −1 .

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Section: Relaxation In the Absence Of Rf Irradiationmentioning

confidence: 99%

Section: Asp/asnmentioning

confidence: 99%

Carbonyl ¹³C transverse relaxation measurements to sample protein backbone dynamics

Mulder

Akke

2003

Magnetic Reson in Chemistry

105

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“…For the J N,CA couplings, numbers in parentheses represent typical ranges in proteins. The transverse relaxation rates R 2 are for a protein with a rotational correlation time s c of 10 ns at a 1 H frequency of 600 MHz a From Wider (1998) b From Cavanagh et al (1996) c From Allard and Härd (1997) be independent of the atom position, so that P(H N ) = P(H a ) = P(H b ). We further used standard values for the J-coupling constants and the transverse relaxation rates R 2 (Table A1) and based the calculations on ideal pathways with transfer delays that optimize the transfer function.…”

Section: Apsy-nmr and Polypeptide Backbone Resonance Assignmentsmentioning

confidence: 99%

APSY-NMR with proteins: practical aspects and backbone assignment

2008

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Automated projection spectroscopy (APSY) is an NMR technique for the recording of discrete sets of projection spectra from higher-dimensional NMR experiments, with automatic identification of the multidimensional chemical shift correlations by the dedicated algorithm GAPRO. This paper presents technical details for optimizing the set-up and the analysis of APSY-NMR experiments with proteins. Since experience so far indicates that the sensitivity for signal detection may become the principal limiting factor for applications with larger proteins or more dilute samples, we performed an APSY-NMR experiment at the limit of sensitivity, and then investigated the effects of varying selected experimental parameters. To obtain the desired reference data, a 4D APSY-HNCOCA experiment with a 12-kDa protein was recorded in 13 min. Based on the analysis of this data set and on general considerations, expressions for the sensitivity of APSY-NMR experiments have been generated to guide the selection of the projection angles, the calculation of the sweep widths, and the choice of other acquisition and processing parameters. In addition, a new peak picking routine and a new validation tool for the final result of the GAPRO spectral analysis are introduced. In continuation of previous reports on the use of APSY-NMR for sequence-specific resonance assignment of proteins, we present the results of a systematic search for suitable combinations of a minimal number of four-and five-dimensional APSY-NMR experiments that can provide the input for algorithms that generate automated protein backbone assignments.

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“…In that case, 13 C directly detected heteronuclear NOE experiments could be advantageously combined to specific COSY-based approaches [29] and/or methyl-TRO-SY experiments [30]. In addition, carbons without directly attached protons usually exhibit advantageous relaxation properties [31,32], and direct detection on carbonyl carbons could help to resolve resonance overlap. Furthermore, it has been shown, more than a decade ago, that homonuclear NOE between adjacent 13 C nuclei could advantageously replace scalar transfer in large proteins [33].…”

Section: Discussionmentioning

confidence: 97%

Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation

Catoire

Zoonens

Heijenoort

et al. 2009

Journal of Magnetic Resonance

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NMR Relaxation Mechanisms for Backbone Carbonyl Carbons in a13C,15N-Labeled Protein

Cited by 29 publications

References 30 publications

Carbonyl ¹³C transverse relaxation measurements to sample protein backbone dynamics

Carbonyl ¹³C transverse relaxation measurements to sample protein backbone dynamics

APSY-NMR with proteins: practical aspects and backbone assignment

Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation

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NMR Relaxation Mechanisms for Backbone Carbonyl Carbons in a13C,15N-Labeled Protein

Cited by 29 publications

References 30 publications

Carbonyl 13C transverse relaxation measurements to sample protein backbone dynamics

Carbonyl 13C transverse relaxation measurements to sample protein backbone dynamics

APSY-NMR with proteins: practical aspects and backbone assignment

Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation

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Product

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Carbonyl ¹³C transverse relaxation measurements to sample protein backbone dynamics

Carbonyl ¹³C transverse relaxation measurements to sample protein backbone dynamics