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Enhanced accuracy of kinetic information from CT-CPMG experiments by transverse rotating-frame spectroscopy

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Abstract

Micro-to-millisecond motions of proteins transmit pivotal signals for protein function. A powerful technique for the measurement of these motions is nuclear magnetic resonance spectroscopy. One of the most widely used methodologies for this purpose is the constant-time Carr–Purcell–Meiboom–Gill (CT-CPMG) relaxation dispersion experiment where kinetic and structural information can be obtained at atomic resolution. Extraction of accurate kinetics determined from CT-CPMG data requires refocusing frequencies that are much larger than the nuclei’s exchange rate between states. We investigated the effect when fast processes are probed by CT-CPMG experiments via simulation and show that if the intrinsic relaxation rate \( \left( {R_{2,0}^{CT - CPMG} } \right) \) is not known a priori the extraction of accurate kinetics is hindered. Errors on the order of 50 % in the exchange rate are attained when processes become fast, but are minimized to 5 % with a priori \( R_{2,0}^{CT - CPMG} \) information. To alleviate this shortcoming, we developed an experimental scheme probing \( R_{2,0}^{CT - CPMG} \) with large amplitude spin-lock fields, which specifically contains the intrinsic proton longitudinal Eigenrelaxation rate. Our approach was validated with ubiquitin and the Oscillatoria agardhii agglutinin (OAA). For OAA, an underestimation of 66 % in the kinetic rates was observed if \( R_{2,0}^{CT - CPMG}\, \)is not included during the analysis of CT-CPMG data and result in incorrect kinetics and imprecise amplitude information. This was overcome by combining CT-CPMG with \( R_{2,0}^{CT - CPMG} \) measured with a high power R experiment. In addition, the measurement of \( R_{2,0}^{CT - CPMG} \) removes the ambiguities in choosing between different models that describe CT-CPMG data.

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Acknowledgments

The work was supported by the Max Planck Society and the EU (ERC Grant Agreement Number 233227) (both to C.G.), and an NIH Grant to A.M.G (GM080642). We are grateful to Dr. Wolfgang Bermel and to Dr. Daniel Mathieu, Bruker Karlsruhe, for uploading the experimental schemes used here to the Bruker standard library (hsqcrexf3gpphtc193d).

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Correspondence to Christian Griesinger or Donghan Lee.

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10858_2013_9769_MOESM1_ESM.pdf

Comparison of different exchange models; Simulations pertaining to measurements at a single spectrometer field strength; Pulse scheme for CT-CPMG to be combined with HEROINE such that the temperatures from both measurements are exactly the same. (PDF 2036 kb)

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Ban, D., Mazur, A., G. Carneiro, M. et al. Enhanced accuracy of kinetic information from CT-CPMG experiments by transverse rotating-frame spectroscopy. J Biomol NMR 57, 73–82 (2013). https://doi.org/10.1007/s10858-013-9769-z

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  • DOI: https://doi.org/10.1007/s10858-013-9769-z

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