A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins.

Autor: Grosskopf JD; Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA., Sidabras JW; Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA., Altenbach C; Department of Chemistry and Biochemistry and Stein Eye Institute, University of California, Los Angeles, California, USA., Anderson JR; Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA., Mett RR; Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA., Strangeway RA; Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA., Hyde JS; Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA., Hubbell WL; Department of Chemistry and Biochemistry and Stein Eye Institute, University of California, Los Angeles, California, USA., Lerch MT; Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Jazyk: angličtina
Zdroj: Protein science : a publication of the Protein Society [Protein Sci] 2024 Dec; Vol. 33 (12), pp. e5220.
DOI: 10.1002/pro.5220
Abstrakt: Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the timescale of ps-μs using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of μs-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and ms-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components (~3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the ms timescale.
(© 2024 The Protein Society.)
Databáze: MEDLINE
Externí odkaz: