Non-equilibrium virus particle dynamics: Microsecond MD simulations of the complete Flock House virus capsid under different conditions.

Flock House virus (FHV) is an animal virus and considered a model system for non-enveloped viruses. It has a small, icosahedral capsid (T=3) and a bipartite positive-sense RNA genome. We present an extensive study of the FHV capsid dynamics from all-atom molecular dynamics simulations of the complete capsid. The simulations explore different biologically relevant conditions (neutral/low pH, with/without RNA in the capsid) using the CHARMM force field. The results show that low pH destabilizes the capsid, causing radial expansion, and RNA stabilizes the capsid. The finding of low pH destabilization is biologically relevant because the capsid is exposed to low pH in the endosome, where conformational changes occur leading to genome release. We also observe structural changes at the fivefold and twofold symmetry axes that likely relate to the externalization of membrane active γ peptides through the fivefold vertex and extrusion of RNA at the twofold axis. Simulations using the Amber force field at neutral pH are also performed and display similar characteristics to the CHARMM simulations.

A favorable path to domain separation in the orange carotenoid protein.

The orange carotenoid protein (OCP) is responsible for nonphotochemical quenching (NPQ) in cyanobacteria, a defense mechanism against potentially damaging effects of excess light conditions. This soluble two-domain protein undergoes profound conformational changes upon photoactivation, involving translocation of the ketocarotenoid inside the cavity followed by domain separation. Domain separation is a critical step in the photocycle of OCP because it exposes the N-terminal domain (NTD) to perform quenching of the phycobilisomes. Many details regarding the mechanism and energetics of OCP domain separation remain unknown. In this work, we apply metadynamics to elucidate the protein rearrangements that lead to the active, domain-separated, form of OCP. We find that translocation of the ketocarotenoid canthaxanthin has a profound effect on the energetic landscape and that domain separation only becomes favorable following translocation. We further explore, characterize, and validate the free energy surface (FES) using equilibrium simulations initiated from different states on the FES. Through pathway optimization methods, we characterize the most probable path to domain separation and reveal the barriers along that pathway. We find that the free energy barriers are relatively small (<5 kcal/mol), but the overall estimated kinetic rate is consistent with experimental measurements (>1 ms). Overall, our results provide detailed information on the requirement for canthaxanthin translocation to precede domain separation and an energetically feasible pathway to dissociation.

Effect of the chemical environment of the DNA guanine quadruplex on the free energy of binding of Na and K ions.

Guanine quadruplex (G-quadruplex) structures play a vital role in stabilizing the DNA genome and in protecting healthy cells from transforming into cancer cells. The structural stability of G-quadruplexes is greatly enhanced by the binding of monovalent cations such as Na+ or K+ into the interior axial channel. We computationally study the free energy of binding of Na+ and K+ ions to two intramolecular G-quadruplexes that differ considerably in their degree of rigidity and the presence or absence of terminal nucleotides. The goal of our study is two-fold. On the one hand, we study the free energy of binding every ion, which complements the experimental findings that report the average free energy for replacing Na+ with K+ ions. On the other hand, we examine the role of the G-quadruplex structure in the binding free energy. In the study, we employ all-atom molecular dynamics simulations and the alchemical transformation method for the computation of the free energies. To compare the cation-dependent contribution to the structural stability of G-quadruplexes, we use a two-step approach to calculate the individual free energy difference ΔG of binding two Na+ and two K+ to two G-quadruplexes: the unimolecular DNA d[T2GT2(G3T)3] (Protein Data Bank ID 2M4P) and the human telomeric DNA d[AGGG(TTAGGG)3] (PDB ID 1KF1). In contrast to the experimental studies that estimate the average free energy of binding, we find a varying difference of approximately 2-9 kcal/mol between the free energy contribution of binding the first and second cation, Na+ or K+. Furthermore, we found that the free energy of binding K+ is not affected by the chemical nature of the two quadruplexes. By contrast, Na+ showed dependency on the G-quadruplex structure; the relatively small size allows Na+ to explore larger configurational space than K+. Numerical results presented here may offer reference values for future design of cationic drug-like ligands that replace the metal ions in G-quadruplexes.

Macroion-Solvent Interactions in Charged Droplets.

Macroion-droplet interactions play a critical role in many settings such as ionization techniques of samples in mass spectrometry analysis and atmospheric aerosols. The droplets under investigation are composed of a polar solvent, primarily water, a charged macroion, and, possibly, buffer ions. We present highlights of our research on the relation between the charge state of a macroion and the droplet morphologies. We have determined that, depending on the charge on the macroion and certain macroscopic properties of the solvent, such as its dielectric constant and surface tension, a droplet may obtain striking conformations such as droplets with extruded tails, "pearl-necklace" conformations, and multipoint "star" shapes. The shapes of the droplet containing the macroion influence the charging mechanism of the macroion in a reciprocal manner. Understanding of the macroion-droplet interactions plays a central role in explaining the origin and the magnitude of the charge in spectra obtained in electrospray ionization mass spectrometry experiments and in controlling the stability of complexes of nucleic acids and proteins in droplets.

Advances in Modeling the Stability of Noncovalent Complexes in Charged Droplets with Applications in Electrospray Ionization-MS Experiments.

Electrospray ionization mass spectrometry is used extensively to measure the equilibrium constant of noncovalent complexes. In this Perspective, we attempt to present an accessible introduction to computational methodologies that can be applied to determine the stability of weak noncovalent complexes in their journey from bulk solution into the gaseous state. We demonstrate the usage of the methods on two typical examples of noncovalent complexes drawn from a broad class of nucleic acids and transient protein complexes found in aqueous droplets. We conclude that this new emerging direction in the use of simulations can lead to estimates of equilibrium constant corrections due to complex dissociation in the carrier droplet and finding of agents that may stabilize the protein interfaces.

Characterization of "Star" Droplet Morphologies Induced by Charged Macromolecules.

"Star" morphologies of charged liquid droplets are distinct droplet conformations that, for a certain charge squared to volume ratio, have lower energy than their spherically shaped analogues. For these shapes to appear, the charge should be carried by a single ionic species. A typical example of a charge carrier that we employ in this study is a fully charged double-stranded oligodeoxynucleotide (dsDNA) in an aqueous and an acetonitrile droplet. We characterize the structure and dynamics of the star-shaped droplets. We find that by increasing the charge squared to volume ratio, the droplet evolves from spherical to "spiky" shapes, by first passing from droplet sizes that undergo enhanced shape fluctuations relative to those of the larger spherical droplets. These fluctuations mark the onset of the instability. We also find that in the spiky droplet, the orientation of the solvent molecules in the first shell about the dsDNA is very close to that in the bulk solution. However, this orientation is substantially different farther away from the dsDNA. With regards to dynamics, the motion of the spikes is reflected in the autocorrelation functions of rotationally invariant order parameters that show a damped oscillator form of decay, indicative of the elastic motion of the spikes. We compare the formation of spikes with that of the ferrofluids and the dielectric materials in an electric field, and we conclude that they represent a different entity that deserves its own characterization. The study provides insight into the manner in which the charge distribution may give rise to well-controlled droplet morphologies and calls for experiments in this direction.

How do non-covalent complexes dissociate in droplets? A case study of the desolvation of dsDNA from a charged aqueous nanodrop.

We present the desolvation mechanism of a double-stranded oligodeoxynucleotide (dsDNA) from an aqueous nanodrop studied by using atomistic molecular dynamics methods. The central theme of this study is the stability of a non-covalently bound complex, in general, and that of a dsDNA in particular, in a droplet environment. Among the factors that may affect the stability of a complex in an evaporating droplet we examine the increase in ion concentration and the distinct droplet morphologies arising from the charge-induced instability. We explore in detail a large set of aqueous nanodrops with excess negative charge, which comprise a dsDNA and Na(+), Cl(-) ions at various concentrations. We find that for a square of the charge to volume ratio above that of the Rayleigh limit the droplet attains distinct "spiky" morphologies that disperse the charge in larger volume relative to that of the spherical drop. Moreover, it is found that it is possible for a non-covalent complex to remain associated in an unstable droplet as long as there is enough solvent to accommodate the instability. In the presence of Na(+) and Cl(-) ions, the Na(+) ions form adducts with the double helical DNA in the minor groove, which help stabilise the duplex state in the gas phase. The negative ions may be released from the droplet. In a DNA-containing droplet with a net charge that is less negative than 50% of the dsDNA charge, the DNA maintains a double-stranded state in the gas phase. Several of our findings are in good agreement with experiments, while the spiky droplet morphology due to the charge-induced instability calls for new experiments. The results shed light on the association properties of complexes of macromolecules in droplet environments, which are critical intermediates in electrospray ionisation experiments.

Effect of counterions on the charging mechanisms of a macromolecule in aqueous nanodrops.

We report the first molecular dynamics study of the effect of counterions on the charging mechanisms of a macromolecule found in an aqueous droplet that contains excess charge. To investigate the principles of the charging mechanisms of a macromolecule in a droplet, we simulate aqueous droplets that contain a poly(ethylene glycol) (PEG) molecule, sodium, and chloride ions. We study the effect of counterions by varying the concentration of the chloride ions and the temperature of the droplets. We find that the size of the droplet from which the macromolecule is released is determined by the competition between the counterions and the macromolecule for capturing the sodium ions. In droplets with radii in the range of 4 nm and smaller, [Na2Cl](+) ion complexes and sodium chloride aggregates are formed. The smaller the droplet the more pronounced is the formation of the NaCl aggregates. At very high temperature, in the larger droplets the Na(+) ions are distributed throughout the entire droplet. Therefore, the sodiated PEG is released with a higher average charge than from droplets with no counterions because it has access to a higher concentration of Na(+) ions. At moderately high temperature, the NaCl aggregates do not affect the final charge state of the macromolecule relative to the no-counterion droplets. We also report that regardless of the concentration of the counterions, the temperature plays a critical role in determining the nature of the droplet shape fluctuations that are responsible for the charging of a macromolecule and its extrusion from a droplet. At high temperature the macromolecule is released by the formation of a Taylor cone that transports ions onto the macromolecule. Differently, at lower temperature the Taylor cones are absent or have subsided. These findings provide insight into the mechanisms that macromolecules acquire their charge in droplets produced in electrospray ionization experiments.