期刊
CHEMPHYSCHEM
卷 21, 期 12, 页码 1224-1229出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cphc.202000312
关键词
DEER; EPR; glass transition of liquid water; protein clustering; rapid freeze quenching
资金
- Korean Visiting Scientist Training Award (KVSTA) from the Korean Health Industry Development Institute
- National Institute of General Medical Sciences of the National Institutes of Health [P41GM103521]
- Intramural Program of the National Institute of Diabetes and Digestive and Kidney Disease
- Office of AIDS Research, NIH [DK029023-19]
- Korea Health Promotion Institute [HI18C2264000019] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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.
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