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

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.

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

“…For instance, the longer the sample spends in a thin film prior to vitrification, the likelier it is to contact the air-water interfaces potentially leading to denaturation 34 or to adopt a preferred orientation, resulting in loss of structural information due to lack of viewing angles 52 . Recent improvements to the support grids and to how the sample is applied have begun to address these issues 33,34,53,54 . Here we describe a different approach, using standard grids rapidly plunging through a sprayed, atomised sample (Figure 1B, Supp Movie 1), faster than 30 ms. We determined a near-atomic resolution structure by single-particle analysis of an apoferritin sample prepared in this way (Figure 4H, I and Supp Figure 4B), which was practically indistinguishable from one obtained by standard preparation (Supp Figure 4C).…”

“…To assess the sample quality obtained by this method we first performed tomographic reconstructions to determine the protein distribution relative to the air-water interfaces [34][35][36] .…”

Structure of the human clamp loader bound to the sliding clamp: a further twist on AAA+ mechanism

“…Alternatively, the open PCNA complex may interact with the airwater interface due to the exposure of hydrophobic residues when the clamp is opened, which may result in aggregation or denaturation (D'Imprima et al 2019;Noble et al 2018). In favor of this latter hypothesis, our cross-linking experiments indicate a significant fraction of the complex is in the open state in solution.…”

Sub-2 Å Resolution Structure Determination Using Single-Particle Cryo-EM at 200 keV

“…The remaining gap might be partially abridged through continued technical developments, such as improvements in detector performance or computational correction of aberrations 9,15-18 but several additional factors currently prevent atomic-resolution cryo-EM SPA of biological specimens. These are largely sample-dependent, and include conformational and/or compositional heterogeneity 19 and the adoption of preferred orientation, as well as specimen denaturation at the hydrophobic *Correspondence to: mherzik@ucsd.edu, glander@scripps.edu air-water interface [20][21][22][23] . Furthermore, high-resolution information of the specimen substantially deteriorates during imaging due to accumulation of beam-induced radiation damage 24 .…”

“…Furthermore, high-resolution information of the specimen substantially deteriorates during imaging due to accumulation of beam-induced radiation damage 24 . Advancements in specimen preparation to minimize air-water interface interactions (e.g., Spotiton 25 , graphene or graphene oxide support films 23,26,27 , streptavidin monolayers 28 ) and image processing strategies to account for conformational dynamics (e.g., focused classification, multi-body refinement 29 , manifold embedding 30 ), are promising avenues that aid in mitigating these oft-encountered issues.…”