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Analysis of Saturation Recovery Amplitudes to Characterize Conformational Exchange in Spin-Labeled Proteins

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Abstract

Analysis of saturation recovery data from spin-labeled proteins is extended to include the amplitudes in addition to the recovery rates for two-site exchange. It is shown that the recovery amplitudes depend strongly on the exchange rate between states as well as their populations and this dependence provides a simple criterion to identify exchange rates in the 10–1000 kHz range. Analysis of experimental SR relaxation curves via the uniform penalty (UPEN) method allows for reliable identification of single, double, or other multiple-component traces, and global fitting of a set of relaxation curves using both relaxation rates and amplitudes determined from the UPEN fits allows for the estimation of exchange rate in the above domain. The theory is tested on simple model systems, and applied to the determination of conformational exchange rates in spin-labeled mutants of T4 Lysozyme and intestinal fatty acid binding protein. Finally, an example of T 1-weighted spectral editing is provided for systems in the slow exchange limit.

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Acknowledgements

This work was supported by National Institute of Health Grants EY05216, T33 EY07026 and 5P41EB001980, and the Jules Stein Professorship endowment. We are grateful to Profs. Sandra and Gareth Eaton (University of Denver) for providing a UPEN analysis program used early on in the study, and for helpful discussions regarding UPEN theory. We also thank Mark Fleissner, Carlos López, and Evan Brooks for graciously providing some of the spin-labeled mutants reported herein.

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Author notes

  1. Zhongyu Yang

    Present address: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58102, USA

Authors and Affiliations

  1. Jules Stein Eye Institute and Department of Chemistry and Biochemistry, UCLA School of Medicine, University of California, Los Angeles, CA, 90095-7008, USA

    Michael D. Bridges, Zhongyu Yang, Christian Altenbach & Wayne L. Hubbell

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  1. Michael D. Bridges
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  2. Zhongyu Yang
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  3. Christian Altenbach
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  4. Wayne L. Hubbell
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Correspondence to Wayne L. Hubbell.

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Bridges, M.D., Yang, Z., Altenbach, C. et al. Analysis of Saturation Recovery Amplitudes to Characterize Conformational Exchange in Spin-Labeled Proteins. Appl Magn Reson 48, 1315–1340 (2017). https://doi.org/10.1007/s00723-017-0936-3

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  • DOI: https://doi.org/10.1007/s00723-017-0936-3

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