Abstract
Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25–50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called “dissipation channel” in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used.
Highlights
Millions of years of evolution have converted viruses into nanomachines optimized to carry out complex functions with a minimalistic structure [1]
We take this result as the upper bound for the resolution of FM-Atomic force microscopy (AFM) that is not reached by the other AFM working modes
According to the data provided by this technique, the parvovirus capsid can be inscribed into a 25 nm diameter sphere and it is formed by 60 structurally equivalent subunits arranged in a simple (T = 1) icosahedral symmetry
Summary
Millions of years of evolution have converted viruses into nanomachines optimized to carry out complex functions with a minimalistic structure [1]. Viruses are masterpieces of nanoengineering with a basic common architecture that consists of the capsid – a protein shell made up of repeating protein subunitswhich packs within it the viral genome Beyond this basic architecture, viruses can have further elaborations such as protein collars, tails, connectors, lipid coats, surface receptors, enzymes, and molecular motors. To the materials engineer or nanotechnologist, viruses are perfectly defined organic nanoparticles which are commonly used as scaffolds or nano-vectors [5] Because their structure can be directed by tailored evolution and their production can be commercially viable, viruses are becoming the basis of whole new approaches to the manufacture of nanomaterials far beyond applications in biology and medicine. While AFM characterization of mechanical properties is usually carried out by nanoindentation [13] in this work we present a new method that allows obtaining, in addition to the topography, spatially resolved maps of flexibility
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