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Carbonyl 13C Transverse Relaxation Measurements to Sample Protein Backbone Dynamics.

Mulder, Frans LU and Akke, Mikael LU (2003) In Magnetic Resonance in Chemistry 41(10). p.853-865
Abstract
Carbonyl 13C relaxation experiments to study protein backbone dynamics have recently been developed. However, the effect of three-bond 13C-13C 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... (More)
Carbonyl 13C relaxation experiments to study protein backbone dynamics have recently been developed. However, the effect of three-bond 13C-13C 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 R1 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 D9k, 13C off-resonance R1 measurements yielded uniform values of R2 along the polypeptide chain, indicating homogeneous chemical shift anisotropies and restricted dynamics on the picosecond to nanosecond time scale. Variation of R2 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 D9k 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. Copyright © 2003 John Wiley & Sons, Ltd. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
backbone, dynamics, calmodulin, calbindin, NMR, C-13 NMR, carbonyl, exchange, CPMG
in
Magnetic Resonance in Chemistry
volume
41
issue
10
pages
853 - 865
publisher
John Wiley & Sons
external identifiers
  • wos:000185662200016
  • scopus:0242299095
ISSN
1097-458X
DOI
language
English
LU publication?
yes
id
95b89231-8a68-4246-9932-0d5457c0cca0 (old id 128096)
date added to LUP
2007-06-29 14:02:31
date last changed
2018-05-29 10:00:48
@article{95b89231-8a68-4246-9932-0d5457c0cca0,
  abstract     = {Carbonyl 13C relaxation experiments to study protein backbone dynamics have recently been developed. However, the effect of three-bond 13C-13C 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 R1 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 D9k, 13C off-resonance R1 measurements yielded uniform values of R2 along the polypeptide chain, indicating homogeneous chemical shift anisotropies and restricted dynamics on the picosecond to nanosecond time scale. Variation of R2 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 D9k 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. Copyright © 2003 John Wiley & Sons, Ltd.},
  author       = {Mulder, Frans and Akke, Mikael},
  issn         = {1097-458X},
  keyword      = {backbone,dynamics,calmodulin,calbindin,NMR,C-13 NMR,carbonyl,exchange,CPMG},
  language     = {eng},
  number       = {10},
  pages        = {853--865},
  publisher    = {John Wiley & Sons},
  series       = {Magnetic Resonance in Chemistry},
  title        = {Carbonyl 13C Transverse Relaxation Measurements to Sample Protein Backbone Dynamics.},
  url          = {http://dx.doi.org/},
  volume       = {41},
  year         = {2003},
}