Submillisecond Freezing Permits Cryoprotectant-Free EPR Double Electron-Electron Resonance Spectroscopy.
Chemphyschem
; 21(12): 1224-1229, 2020 06 16.
Article
en En
| MEDLINE
| ID: mdl-32383308
ABSTRACT
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 (77â
K or less) to prolong the phase memory relaxation time (Tm ) 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 Tm ) during relatively slow flash freezing in liquid N2 . 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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Virales
/
Espectroscopía de Resonancia por Spin del Electrón
Límite:
Animals
Idioma:
En
Revista:
Chemphyschem
Asunto de la revista:
BIOFISICA
/
QUIMICA
Año:
2020
Tipo del documento:
Article
País de afiliación:
Estados Unidos