A Microfluidic Rapid Freeze Quench Apparatus for High Field EPR Measurements

Abstract

Rapid freeze quench (RFQ) EPR is a well-established technique for trapping reaction intermediates. A major difficulty in using commercial RFQ-EPR combined with standard X-band EPR spectroscopy is the relatively large amount of sample needed for each time point, and the associated amount that is wasted in the dead volume of the tubes and mixer. This is particularly prohibitive when one would like to couple RFQ with high resolution EPR techniques such as ENDOR (electron-nuclear double resonance) and DEER (electron-electron double resonance) that provide electron-nuclear and electron-electron distances and are less sensitive than standard continuous wave EPR. We have developed a dedicated microfluidic RFQ (RFQ) apparatus for W-band measurements, optimized for the small W-band sample size and a minimal sample amount for a series of ∼7 time points collected in triplicates (∼200 l of 0.03-0.1 mM labeled protein). The mixer is based on a recent published design[1] with a modified sample ejection system and cold trap. It current time window is 5-90 msec and its performance has been demonstrated on the reduction of nitroxide with dithionite[2]. The current state of the RFQ apparatus is demonstrated on : (i) the ATPase activity of an RNA helicase. Here Mg2+ was substituted with Mn2+ and the 31P ENDOR spectrum was recorded. The ADP and ATP spectra have significantly different lineshapes and can therefore be used to probe the hydrolysis state. (ii) Conformational changes induced in a protein through ligand binding as detected by DEER. 1. Egawa T, Durand JL, Hayden EY, Rousseau DL, Yeh S-R. Anal Chem. 2009;81(4):1622-7. 2. R. Kaufmann, D. Goldfarb.. J Magn Reson. 2013;230:220-6.