Medical College of Wisconsin
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A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins. Protein Sci 2024 Dec;33(12):e5220

Date

11/20/2024

Pubmed ID

39565088

Pubmed Central ID

PMC11577460

DOI

10.1002/pro.5220

Scopus ID

2-s2.0-85210009782 (requires institutional sign-in at Scopus site)   3 Citations

Abstract

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.

Author List

Grosskopf JD, Sidabras JW, Altenbach C, Anderson JR, Mett RR, Strangeway RA, Hyde JS, Hubbell WL, Lerch MT

Authors

Michael Lerch PhD Associate Professor in the Biophysics department at Medical College of Wisconsin
Jason W. Sidabras PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Bacteriophage T4
Electron Spin Resonance Spectroscopy
Muramidase
Pressure
Protein Conformation
Spin Labels