Multifrequency Force Microscopy of Helical Protein Assembly on a Virus

Abstract

High-resolution microscopy techniques have been extensively used to investigate the structure of soft, biological matter at the nanoscale, from very thin membranes to small objects, like viruses. Electron microscopy techniques allow for obtaining extraordinary resolution by averaging signals from multiple identical structures. In contrast, atomic force microscopy (AFM) collects data from single entities. Here, it is possible to finely modulate the interaction with the samples, in order to be sensitive to their top surface, avoiding mechanical deformations. However, most biological surfaces are highly curved, such as fibers or tubes, and ultimate details of their surface are in the vicinity of steep height variations. This limits lateral resolution, even when sharp probes are used. We overcome this problem by using multifrequency force microscopy on a textbook example, the Tobacco Mosaic Virus (TMV). We achieved unprecedented resolution in local maps of amplitude and phase shift of the second excited mode, recorded together with sample topography. Our data, which combine multifrequency imaging and Fourier analysis, confirm the structure deduced from averaging techniques (XRD, cryoEM) for surface features of single virus particles, down to the helical pitch of the coat protein subunits, 2.3 nm. Remarkably, multifrequency AFM images do not require any image postprocessing.

Nanoscale wetting of viruses by ionic liquids

“…1 exhibits an apparent height of ~ 14±0.3 nm. This value is typical for TMV on hydrophilic substrates [39], and differs from the theoretical height of 18 nm, which is found for TMV particles by non-contact AFM on more hydrophobic substrates such as graphite [31] and on alkyl monolayers [40]. The flattening observed on hydrophilic substrates is a consequence of the strong virion-substrate interactions.…”

“…Some of them are 700 nm-1000 nm long due to the formation of headto-tail aggregates of two to three virions in suspension [38]. Particles shorter than 300 nm are protein aggregates, including single disks and/or vertically stacked disks of TMV coat proteins [39]. The profile of the virion in Fig.…”

Nanoscale Wetting of Single Viruses

“…Images were collected at increasing humidity conditions, i.e., at RH = 56% ( Figure 2 a,b), 76% ( Figure 2 c,d), and >100% ( Figure 2 e,f) (see Materials and Methods). Multifrequency images were recorded in the net attractive tip-sample interaction regime for the first fundamental mode [ 29 , 30 ], as inferred from the phase shift of the low frequency excitation (Φ 1 ), which always exceeded 90° [ 12 ]. The sensitivity of the second mode leads to a much higher contrast than standard measurements based on the fundamental mode (see Figure S2 of the Supplementary Materials ) and allows for clear identification of water morphologies for sizes above ~0 nm.…”

“…The exceptional stability of TMV particles makes them optimal candidates for experiments that need to be performed in air. The characteristic tubular shape with a typical length of ~300 nm and a height of ~14 nm is preserved (the diameter of 18 nm found in bulk virus samples is reduced by radial deformation upon adsorption on surfaces) [ 12 , 13 ]. In TMV, the high mechanical and chemical stability [ 14 , 15 ] is related to the need for this plant virus to “survive” in air and in soil, under highly variable and often adverse environmental conditions [ 16 ].…”

“…In this work, we present standard AFM topographic images correlated with phase shift images obtained with the second excited mode (Φ 2 ), which were collected simultaneously with the topography. Even with optimized multifrequency imaging conditions [ 12 ], the quantification of the amount of water adsorbed on the surface of hydrated TMV particles remains challenging and can be barely deduced from a statistical analysis of many topographic profiles collected at various humidity levels. Here we show that dynamic force spectroscopy [ 23 , 24 ] allows detecting a thin water layer, ~1 nm thick, on the surface of single TMV particles at ambient and low RH < 60%.…”