An improvement to capture and visualize the protein conformational ensembles in double electron-electron resonance measurements.

Abstract

Over the past decades, pulsed ESR double electron-electron resonance (DEER) has emerged as an effective biophysical tool for revealing protein conformational ensembles. With a collection of numerous interspin distances by DEER, they can be utilized to reveal the dynamic structural ensembles as opposed to static structures from other techniques. In a continuing effort to improve the technique, we present a combined approach of experimental and simulation advances that allows one to capture and visualize protein conformational ensembles in the absence of cryoprotectant additives. First, we present a rapid freezing method using a flexible fused silica microcapillary, enabling DEER measurement without the use of cryoprotectants. Second, we present a Monte Carlo based algorithm that allows one to efficiently utilize the sparse DEER data for deriving ensemble structures of a target protein. The Bid protein, a highly glycerol-sensitive protein, is used as a model. We show that our rapid-freezing DEER method can capture the Bid ensembles in the absence of glycerol, which was not possible with the conventional DEER setup. With the newly developed algorithm, we can efficiently visualize the Bid conformational ensembles, saving a lot for performing advanced structural modeling. Our combined method advances DEER for capturing and visualizing protein conformational ensembles.