Protein denaturation at the air-water interface and how to prevent it.

Edoardo D'Imprima,Mirko Joppe,Martin Grininger,Davide Floris,Ricardo Sánchez,Werner Kühlbrandt

doi:10.7554/elife.42747

Edoardo D'Imprima, Mirko Joppe + Show 4 more

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https://doi.org/10.7554/elife.42747

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Abstract

Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

Highlights

In the short time since the resolution revolution (Kuhlbrandt, 2014), single-particle electron cryomicroscopy has developed into a main technique for high resolution structure determination of proteins (Bai et al, 2015a)
The fatty acid synthase (FAS) complex used for cryo-EM data collection was pure and homogeneous, as shown by size exclusion chromatography, SDS-polyacrylamide gel electrophoresis and blue-native polyacrylamide gel electrophoresis (Figure 1—figure supplement 1A–C)
Thermal shift assays indicated that the complex was stable (Figure 1—figure supplement 1D), and at 1500–3000 mU/mg it was enzymatically fully active (Fichtlscherer et al, 2000; Oesterhelt et al, 1969; Wieland et al, 1979)

Summary

Introduction

In the short time since the resolution revolution (Kuhlbrandt, 2014), single-particle electron cryomicroscopy (cryo-EM) has developed into a main technique for high resolution structure determination of proteins (Bai et al, 2015a). To achieve high contrast and high resolution in cryo-EM, a small volume of protein solution is applied to an EM support grid (Cheng et al, 2015; Passmore and Russo, 2016) and blotted before vitrification by plunge-freezing in liquid ethane (Dubochet et al, 1988; McDowall et al, 1983). During this process, the protein is inevitably exposed to the atmosphere at a high surface-to-volume ratio. Recent studies (Glaeser, 2018; Glaeser and Han, 2017; Han et al, 2017) have drawn attention to the effects of the air water interface on proteins in solution, in particular on their integrity and orientation on cryoEM grids

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