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Submillisecond Freezing Permits Cryoprotectant‐Free EPR Double Electron−Electron Resonance Spectroscopy
Author(s) -
Schmidt Thomas,
Jeon Jaekyun,
Okuno Yusuke,
Chiliveri Sai C.,
Clore G. Marius
Publication year - 2020
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.202000312
Subject(s) - cryoprotectant , electron paramagnetic resonance , chemistry , site directed spin labeling , liquid nitrogen , analytical chemistry (journal) , lipid bilayer , crystallography , biophysics , chromatography , nuclear magnetic resonance , membrane , biochemistry , cryopreservation , embryo , physics , organic chemistry , biology , microbiology and biotechnology
Double electron‐electron resonance (DEER) EPR spectroscopy is a powerful method for obtaining distance distributions between pairs of engineered nitroxide spin‐labels in proteins and other biological macromolecules. These measurements require the use of cryogenic temperatures (77 K or less) to prolong the phase memory relaxation time ( T m ) sufficiently to enable detection of a DEER echo curve. Generally, a cryoprotectant such as glycerol is added to protein samples to facilitate glass formation and avoid protein clustering (which can result in a large decrease in T m ) during relatively slow flash freezing in liquid N 2 . However, cryoprotectants are osmolytes and can influence protein folding/unfolding equilibria, as well as species populations in weak multimeric systems. Here we show that submillisecond rapid freezing, achieved by high velocity spraying of the sample onto a rapidly spinning, liquid nitrogen cooled copper disc obviates the requirement for cryoprotectants and permits high quality DEER data to be obtained in absence of glycerol. We demonstrate this approach on five different protein systems: protein A, the metastable drkN SH3 domain, urea‐unfolded drkN SH3, HIV‐1 reverse transcriptase, and the transmembrane domain of HIV‐1 gp41 in lipid bicelles.