Pedro J. de Pablo

Manipulation of the mechanical properties of a virus by protein engineering

In a previous study, we showed that the DNA molecule within a spherical virus (the minute virus of mice) plays an architectural role by anisotropically increasing the mechanical stiffness of the virus. A finite element model predicted that this mechanical reinforcement is a consequence of the interaction between crystallographically visible, short DNA patches and the inner capsid wall. We have now tested this model by using protein engineering. Selected amino acid side chains have been truncated to specifically remove major interactions between the capsid and the visible DNA patches, and the effect of the mutations on the stiffness of virus particles has been measured using atomic force microscopy. The mutations do not affect the stiffness of the empty capsid; however, they significantly reduce the difference in stiffness between the DNA-filled virion and the empty capsid. The results (i) reveal that intermolecular interactions between individual chemical groups contribute to the mechanical properties of a supramolecular assembly and (ii) identify specific protein–DNA interactions as the origin of the anisotropic increase in the rigidity of a virus. This study also demonstrates that it is possible to control the mechanical properties of a protein nanoparticle by the rational application of protein engineering based on a mechanical model.

Origins of phase contrast in the atomic force microscope in liquids

We study the physical origins of phase contrast in dynamic atomic force microscopy (dAFM) in liquids where low-stiffness microcantilever probes are often used for nanoscale imaging of soft biological samples with gentle forces. Under these conditions, we show that the phase contrast derives primarily from a unique energy flow channel that opens up in liquids due to the momentary excitation of higher eigenmodes. Contrary to the common assumption, phase-contrast images in liquids using soft microcantilevers are often maps of short-range conservative interactions, such as local elastic response, rather than tip-sample dissipation. The theory is used to demonstrate variations in local elasticity of purple membrane and bacteriophage ϕ29 virions in buffer solutions using the phase-contrast images.

The Role of Capsid Maturation on Adenovirus Priming for Sequential Uncoating

Background: Adenovirus proteolytic maturation is required for correct uncoating in the cell. Results: Maturation makes the virion metastable and facilitates penton and peripheral core protein release, as well as cooperative genome ejection. Conclusion: Precursor proteins act as scaffolds favoring assembly. Maturation primes adenovirus for uncoating. Significance: Identifying the molecular determinants of virus stability and uncoating is key to understanding the infectious cycle. Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus.

Direct measurement of phage phi29 stiffness provides evidence of internal pressure.

Using AFM nanoindentation experiments, DNA-full phi29 phage capsids are shown to be stiffer than when empty. The presence of counterions softens full viruses in a reversible manner, indicating that pressure originates from the confined DNA. A finite element analysis of the experiments provides an estimate of the pressure of ∼40 atm inside the capsid, which is similar to theoretical predictions.

Unmasking imaging forces on soft biological samples in liquids when using dynamic atomic force microscopy: a case study on viral capsids.

Dynamic atomic force microscopy is widely used for the imaging of soft biological materials in liquid environments; yet very little is known about the peak forces exerted by the oscillating probe tapping on the sample in liquid environments. In this article, we combine theory and experiments in liquid on virus capsids to propose scaling laws for peak interaction forces exerted on soft samples in liquid environments. We demonstrate how these laws can be used to choose probes and operating conditions to minimize imaging forces and thereby robustly image fragile biological samples.

Mechanical elasticity as a physical signature of conformational dynamics in a virus particle

In this study we test the hypothesis that mechanically elastic regions in a virus particle (or large biomolecular complex) must coincide with conformationally dynamic regions, because both properties are intrinsically correlated. Hypothesis-derived predictions were subjected to verification by using 19 variants of the minute virus of mice capsid. The structural modifications in these variants reduced, preserved, or restored the conformational dynamism of regions surrounding capsid pores that are involved in molecular translocation events required for virus infectivity. The mechanical elasticity of the modified capsids was analyzed by atomic force microscopy, and the results corroborated every prediction tested: Any mutation (or chemical cross-linking) that impaired a conformational rearrangement of the pore regions increased their mechanical stiffness. On the contrary, any mutation that preserved the dynamics of the pore regions also preserved their elasticity. Moreover, any pseudo-reversion that restored the dynamics of the pore regions (lost through previous mutation) also restored their elasticity. Finally, no correlation was observed between dynamics of the pore regions and mechanical elasticity of other capsid regions. This study (i) corroborates the hypothesis that local mechanical elasticity and conformational dynamics in a viral particle are intrinsically correlated; (ii) proposes that determination by atomic force microscopy of local mechanical elasticity, combined with mutational analysis, may be used to identify and study conformationally dynamic regions in virus particles and large biomolecular complexes; (iii) supports a connection between mechanical properties and biological function in a virus; (iv) shows that viral capsids can be greatly stiffened by protein engineering for nanotechnological applications.

Synthesis of designed conductive one-dimensional coordination polymers of Ni(II) with 6-mercaptopurine and 6-thioguanine.

Calculations performed with the goal of designing suitable electrical conductive [M(6-MP)(2)](n) (M = transition metal, 6-MP = 6-mercaptopurinato) one-dimensional coordination polymers suggested that metal ions such as Ni(II) could provide suitable materials. In this work, direct hydrothermal reactions between 6-mercaptopurine (6-MPH) and the analogous 6-thioguanine (6-ThioGH) with NiSO(4).6H(2)O yield the compounds [Ni(6-MP)(2)](n).2nH(2)O [1] and [Ni(6-ThioG)(2)](n).2nH(2)O [2]. The X-ray structures confirm that both compounds present similar structures based on one-dimensional chains in which the deprotonated nucleobases act as the bridging ligands connecting the metal ions by short distances. Electrical measurements at room temperature confirm the conductor character of both coordination polymers. The small differences found in these measurements have been rationalized with the help of density functional theory calculations. Preliminary adsorption studies on surfaces for 1 have allowed characterization of single chains on mica and graphite. The results obtained suggest the potential use of coordination polymers on nanomaterials for molecular electronics.

MMX polymer chains on surfaces

Fibres of [Ru(2)Br(micro-O(2)CEt)4]n polymer have been isolated on different surfaces under specific conditions, and morphologically characterised by AFM and STM, showing an unexpected helical internal structure.

Fluorescence Tracking of Genome Release during Mechanical Unpacking of Single Viruses

Viruses package their genome in a robust protein coat to protect it during transmission between cells and organisms. In a reaction termed uncoating, the virus is progressively weakened during entry into cells. At the end of the uncoating process the genome separates, becomes transcriptionally active, and initiates the production of progeny. Here, we triggered the disruption of single human adenovirus capsids with atomic force microscopy and followed genome exposure by single-molecule fluorescence microscopy. This method allowed the comparison of immature (noninfectious) and mature (infectious) adenovirus particles. We observed two condensation states of the fluorescently labeled genome, a feature of the virus that may be related to infectivity. Beyond tracking the unpacking of virus genomes, this approach may find application in testing the cargo release of bioinspired delivery vehicles.

Mechanical Disassembly of Single Virus Particles Reveals Kinetic Intermediates Predicted by Theory

New experimental approaches are required to detect the elusive transient intermediates predicted by simulations of virus assembly or disassembly. Here, an atomic force microscope (AFM) was used to mechanically induce partial disassembly of single icosahedral T=1 capsids and virions of the minute virus of mice. The kinetic intermediates formed were imaged by AFM. The results revealed that induced disassembly of single minute-virus-of-mice particles is frequently initiated by loss of one of the 20 equivalent capsomers (trimers of capsid protein subunits) leading to a stable, nearly complete particle that does not readily lose further capsomers. With lower frequency, a fairly stable, three-fourths-complete capsid lacking one pentamer of capsomers and a free, stable pentamer were obtained. The intermediates most frequently identified (capsids missing one capsomer, capsids missing one pentamer of capsomers, and free pentamers of capsomers) had been predicted in theoretical studies of reversible capsid assembly based on thermodynamic-kinetic models, molecular dynamics, or oligomerization energies. We conclude that mechanical manipulation and imaging of simple virus particles by AFM can be used to experimentally identify kinetic intermediates predicted by simulations of assembly or disassembly.