Simple assay for adsorption of proteins to the air–water interface

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Highlights

  • Rapid method to detect whether samples form denatured-protein monolayers.

  • Uses 10 µL aliquots at any chosen protein concentration.

  • Provides estimates for the speed with which adsorption occurs.

  • Method is suitable to screen for the effects of additives.

  • Facilitates the screening of conditions used to prepare grids for cryo-EM.

Abstract

A rapid assay is described, based upon the Marangoni effect, which detects the formation of a denatured-protein film at the air–water interface (AWI) of aqueous samples. This assay requires no more than a 20 µL aliquot of sample, at a protein concentration of no more than1 mg/ml, and it can be performed with any buffer that is used to prepare grids for electron cryo-microscopy (cryo-EM). In addition, this assay provides an easy way to estimate the rate at which a given protein forms such a film at the AWI. Use of this assay is suggested as a way to pre-screen the effect of various additives and chemical modifications that one might use to optimize the preparation of grids, although the final proof of optimization still requires further screening of grids in the electron microscope. In those cases when the assay establishes that a given protein does form a sacrificial, denatured-protein monolayer, it is suggested that subsequent optimization strategies might focus on discovering how to improve the adsorption of native proteins onto that monolayer, rather than to prevent its formation. A second alternative might be to bind such proteins to the surface of rationally designed affinity grids, in order to prevent their diffusion to, and unwanted interaction with, the AWI.

Introduction

With the emergence of single-particle electron cryo-microscopy (cryo-EM) as a popular technology for determining the structures of biological macromolecules (2021, xxxx), the ease with which proteins adsorb to the air–water interface (AWI) has become an issue of major concern (Glaeser, 2021). As background, cryo-EM uses specimens whose ideal thickness is comparable to the size of the macromolecular particles themselves, and in any case not thicker than about 100 nm. As a result, the time required for particles to diffuse to the AWI – after formation of a suitably thin specimen – is on the scale of 1 ms or less (Taylor and Glaeser, 2008, Naydenova and Russo, 2017). As a consequence, it is not possible for freely diffusing particles to avoid colliding many times with the AWI prior to vitrification.

Unfortunately, there is a high risk of denaturation upon contact, and thus touching the AWI is a very dangerous thing for proteins to do. Nevertheless, as many as 10% to 25% of samples can be prepared for cryo-EM with little difficulty, and preparation of many other samples, which may initially prove to be challenging, eventually succeeds after extensive, particle-specific optimization of the buffer and other conditions (Carragher et al., 2019, Drulyte et al., 2018, Klebl et al., 2020). One way to reconcile the frequent success with the obvious hazard of grid preparation might be to hypothesize that a sacrificial, denatured-protein skin almost always forms at, and passivates, the AWI, to which native proteins can be bound – at least under optimized conditions – in a structure-friendly way (Glaeser, 2021).

The literature provides surprisingly little guidance as to whether a newly investigated protein can be expected to denature upon diffusing to and touching the AWI. Although the study of protein adsorption to the AWI is quite mature, the number of test specimens that have historically been characterized is, in fact, rather limited relative to the number and variety of proteins now being studied by cryo-EM. Furthermore, the characterization of adsorption and denaturation of proteins at the AWI has historically involved a lengthy study in its own right, rather than employing a simple assay that could be used as part of a larger study. As a result, there is a need to develop fast and easy methods that might be used to assist optimization of the conditions used to prepare a new type of sample for cryo-EM.

Here we introduce an assay that uses the Marangoni effect (see section 20.4 of (Rapp, 2017) for background) to detect the modification of the AWI that occurs when proteins are adsorbed to and denatured at the interface. Briefly, the Marangoni effect refers to phenomena involving mass transfer of a liquid that is driven by a gradient of the surface tension. The breakup of an existing, thin film of water into thicker puddles, with surrounding areas that appear to be almost dry, when small amounts of waste alcohol are thrown into a wet sink, is an example of the Marangoni effect that will be familiar to most biochemists. Another example that is familiar to many, not just biochemist!, is the “tears of wine” phenomenon, described on many internet web sites.

Each test in our assay can be done in 10 min or less and requires only 10 µL or less of sample. Using this assay, we have observed that most proteins quickly modify the AWI, many at concentrations as low as 0.15–0.25 mg/mL. Two proteins, however, one of which has already been shown by cryo-EM to bind to the AWI with a strongly preferred orientation, produced relatively little effect, even at concentrations as high as 1 mg/mL.

We propose that this assay can, in the first instance, reduce or even remove uncertainly about whether a denatured-protein monolayer forms at the AWI, under the same conditions that are used when preparing grids for cryo-EM. This assay also provides an easy way to estimate the speed at which such denatured-protein monolayers are formed. Going beyond that, the assay provides a useful way to screen for additives or chemical treatments that might improve the preparation of EM samples by preventing adsorption of proteins to the AWI. The final proof of optimization will nevertheless remain with subsequently screening grids in the electron microscope.

Section snippets

Reagents

A total of 13 soluble-protein samples were used as test specimens during the development and evaluation of the assay described here. The names of 11 of the soluble proteins, and the buffers in which they were tested, are given in Table. 1. Ferritin, carbonic anhydrase, lysozyme, cytochrome C, catalase, bovine serum albumin, and avidin were purchased from Sigma Aldrich, while streptavidin was purchased from New England Biolabs. Three additional soluble-protein samples were gifts of colleagues at

Methods

The assay is performed by first applying 10 µL of an aqueous sample onto a hydrophilic surface in order to form a puddle that is ∼1 cm in diameter. The initial adsorption of proteins to the AWI of this small puddle is expected to closely mimic what happens prior to blotting, when a smaller volume of sample, for example 3 μL, is first deposited onto the correspondingly smaller area of an EM grid. We use an ∼1-inch square piece of freshly cleaved mica as the hydrophilic substrate, which produces

Results

When a small drop of nonafluorobutyl methyl ether (NFBME) is spotted onto the AWI, near to the middle of a puddle of buffer that is free of surfactant, this immediately causes the buffer to pull away from the point where the NFBME was applied, as can be seen by comparing the image shown in Fig. 2F to the image shown in Fig. 2A. A realtime video, for which the sample was lysozyme at a concentration of only 0.1 mg/mL, is included as Supplemental material in order to illustrate the speed with

Discussion

The assay introduced here provides a fast and inexpensive way (A) to determine whether a specimen of interest makes a denatured-protein film at the air–water interface (AWI) and, if it does, (B) to estimate how rapidly such a film is formed. The assay is based upon the fact, demonstrated here, that local application of a water-insoluble, volatile surfactant causes a puddle of buffer to retract from the point of application, whereas moderately high concentrations of soluble proteins suppress or

CRediT authorship contribution statement

Bong-Gyoon Han: Conceptualization, Methodology, Data curation, Visualization, Reviewing and Editing. Robert M. Glaeser: Conceptualization, Supervision, Methodology, Writing - Original draft preparation, Writing - Reviewing and Editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work has been supported in part through funds awarded by Genentech, and we thank Dr. Chris Arthur and others at Genentech for their interest in our work on protein adsorption to the AWI. We thank the following colleagues at the University of California for donating samples to use in the development and testing of the assay described here: samples of rubisco were provided by Luke Oltrogge in the laboratory of Prof. David Savage; samples of FIP200:13 and ATG9 were provided by Adam Yokum in

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