Opinion: hazards faced by macromolecules when confined to thin aqueous films

Abstract

Samples prepared for single-particle electron cryo-microscopy (cryo-EM) necessarily have a very high surface-to-volume ratio during the short period of time between thinning and vitrification. During this time, there is an obvious risk that macromolecules of interest may adsorb to the air-water interface with a preferred orientation, or that they may even become partially or fully unfolded at the interface. In addition, adsorption of macromolecules to an air-water interface may occur even before thinning. This paper addresses the question whether currently used methods of sample preparation might be improved if one could avoid such interfacial interactions. One possible way to do so might be to preemptively form a surfactant monolayer over the air-water interfaces, to serve as a structure-friendly slide and coverslip. An alternative is to immobilize particles of interest by binding them to some type of support film, which-to continue using the analogy-thus serves as a slide. In this case, the goal is not only to prevent the particles of interest from diffusing into contact with the air-water interface but also to increase the number of particles seen in each image. In this direction, it is natural to think of developing various types of affinity grids as structure-friendly alternatives to thin carbon films. Perhaps ironically, if precautions are not taken against adsorption of particles to air-water interfaces, sacrificial monolayers of denatured protein may take the roles of slide, coverslip, or even both.

Keywords: Air–water interface; Cryo-EM; Protein denaturation; Sample preparation.

Fig. 1

Cartoon representation of an idealized cryo-EM specimen. Note that the particle (red), the hole in the carbon film (brown), and the thin film of vitreous ice (blue) are not drawn to scale. The main point of this cartoon is to illustrate the goal that the particle should be surrounded on all sides by vitrified buffer, and—in particular—the particle should not touch the air–water interface

Fig. 2

Schematic diagram of the technique introduced by Trurnit (1960) for the quantitative transfer of proteins to the air–water interface of a Langmuir trough. Protein solutions are delivered by pipette to the top of a clean glass rod, of length l, after which they flow down as a liquid curtain of thickness d, which can be as thin as 10 µm. Protein molecules that diffuse to, and adsorb to, the air–water interface, as the sample flows down the surface of the glass rod, are necessarily delivered to the surface of the trough solution. Reproduced with permission from Trurnit (1960)

Fig. 3

Comparison of schematic free-energy “landscapes” for unfolding of proteins at the air–water interface (lower curve) versus when in bulk solution (upper curve). The native state, “N” is indicated at the beginning of the unfolding reaction, and the unfolded state, “U” is indicated at the end of the reaction. The arrow on the right-hand side indicates the expectation that the free energy is very favorable for transfer of an unfolded protein from bulk water (upper curve) to the air–water interface (lower curve). As is argued in the text, this process is nevertheless expected to be rate limited by the low frequency with which unfolded species form in bulk water. As is further argued in the text, however, contact between a protein and the air–water interface can lead to spontaneous unfolding, without any significant activation barriers for completion of the reaction

Fig. 4

Two examples of unfolded lysozyme structures obtained in molecular dynamics (MD) simulations performed by Raffaini and Ganazzoli (2010). Although the substrate was graphite in these simulations, our opinion is that similar results are to be expected for the air–water interface. It is worth noting that these simulations indicate that an ensemble of unfolded structures is likely to be formed, with the orientation of the protein at the time of initial contact playing an important role in determining the final, unfolded structure. Reproduced with permission

Fig. 5

Cartoon to show immobilized particles with the air–water interface well above the particle (A) and with the interface touching the particle (B). In the ideal case, like that shown schematically in A, particles bound to a structure-friendly support film (slide) are not at risk of becoming denatured at the air–water interface as long as the surrounding layer of buffer remains thicker than the size of the particle. As the layer of buffer becomes thinner and thinner, however, the air–water interface must eventually touch the particle, as shown in B