Constriction of Actin Rings by Passive Crosslinkers.

In many organisms, cell division is driven by the constriction of a cytokinetic ring, which consists of actin filaments and crosslinking proteins. While it has long been believed that the constriction is driven by motor proteins, it has recently been discovered that passive crosslinkers that do not turn over fuel are able to generate enough force to constrict actin filament rings. To study the ring constriction dynamics, we develop a model that includes the driving force of crosslinker condensation and the opposing forces of friction and filament bending. We analyze the constriction force as a function of ring topology and crosslinker concentration, and predict forces that are sufficient to constrict an unadorned plasma membrane. Our model also predicts that actin-filament sliding arises from an interplay between filament rotation and crosslinker hopping, producing frictional forces that are low compared with those of crosslinker-mediated microtubule sliding.

Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions.

DNA-PAINT based single-particle tracking (DNA-PAINT-SPT) has recently significantly enhanced observation times in in vitro SPT experiments by overcoming the constraints of fluorophore photobleaching. However, with the reported implementation, only a single target can be imaged and the technique cannot be applied straight to live cell imaging. Here we report on leveraging this technique from a proof-of-principle implementation to a useful tool for the SPT community by introducing simultaneous live cell dual-color DNA-PAINT-SPT for quantifying protein dimerization and tracking proteins in living cell membranes, demonstrating its improved performance over single-dye SPT.

Simulations of DNA-Origami Self-Assembly Reveal Design-Dependent Nucleation Barriers.

Nucleation is the rate-determining step in the kinetics of many self-assembly processes. However, the importance of nucleation in the kinetics of DNA-origami self-assembly, which involves both the binding of staple strands and the folding of the scaffold strand, is unclear. Here, using Monte Carlo simulations of a lattice model of DNA origami, we find that some, but not all, designs can have a nucleation barrier and that this barrier disappears at lower temperatures, rationalizing the success of isothermal assembly. We show that the height of the nucleation barrier depends primarily on the coaxial stacking of staples that are adjacent on the same helix, a parameter that can be modified with staple design. Creating a nucleation barrier to DNA-origami assembly could be useful in optimizing assembly times and yields, while eliminating the barrier may allow for fast molecular sensors that can assemble/disassemble without hysteresis in response to changes in the environment.

Lattice models and Monte Carlo methods for simulating DNA origami self-assembly.

The optimal design of DNA origami systems that assemble rapidly and robustly is hampered by the lack of a model for self-assembly that is sufficiently detailed yet computationally tractable. Here, we propose a model for DNA origami that strikes a balance between these two criteria by representing these systems on a lattice at the level of binding domains. The free energy of hybridization between individual binding domains is estimated with a nearest-neighbour model. Double helical segments are treated as being rigid, but we allow flexibility at points where the backbone of one of the strands is interrupted, which provides a reasonably realistic representation of partially and fully assembled states. Particular attention is paid to the constraints imposed by the double helical twist, as they determine where strand crossovers between adjacent helices can occur. To improve the efficiency of sampling configuration space, we develop Monte Carlo methods for sampling scaffold conformations in near-assembled states, and we carry out simulations in the grand canonical ensemble, enabling us to avoid considering states with unbound staples. We demonstrate that our model can quickly sample assembled configurations of a small origami design previously studied with the oxDNA model, as well as a design with staples that span longer segments of the scaffold. The sampling ability of our method should allow for good statistics to be obtained when studying the assembly pathways and is suited to investigating, in particular, the effects of design and assembly conditions on these pathways and their resulting final assembled structures.

Free energies of solvation in the context of protein folding: Implications for implicit and explicit solvent models.

Implicit solvent models for biomolecular simulations have been developed to use in place of more expensive explicit models; however, these models make many assumptions and approximations that are likely to affect accuracy. Here, the changes in free energies of solvation upon folding ΔΔGsolv of several fast folding proteins are calculated from previously run µs-ms simulations with a number of implicit solvent models and compared to the values needed to be consistent with the explicit solvent model used in the simulations. In the majority of cases, there is a significant and substantial difference between the ΔΔGsolv values calculated from the two approaches that is robust to the details of the calculations. These differences could only be remedied by selecting values for the model parameters-the internal dielectric constant for the polar term and the surface tension coefficient for the nonpolar term-that were system-specific or physically unrealistic. We discuss the potential implications of our findings for both implicit and explicit solvent simulations. © 2015 Wiley Periodicals, Inc.

Promiscuity as a functional trait: intrinsically disordered regions as central players of interactomes.

Because of their pervasiveness in eukaryotic genomes and their unique properties, understanding the role that ID (intrinsically disordered) regions in proteins play in the interactome is essential for gaining a better understanding of the network. Especially critical in determining this role is their ability to bind more than one partner using the same region. Studies have revealed that proteins containing ID regions tend to take a central role in protein interaction networks; specifically, they act as hubs, interacting with multiple different partners across time and space, allowing for the co-ordination of many cellular activities. There appear to be three different modules within ID regions responsible for their functionally promiscuous behaviour: MoRFs (molecular recognition features), SLiMs (small linear motifs) and LCRs (low complexity regions). These regions allow for functionality such as engaging in the formation of dynamic heteromeric structures which can serve to increase local activity of an enzyme or store a collection of functionally related molecules for later use. However, the use of promiscuity does not come without a cost: a number of diseases that have been associated with ID-containing proteins seem to be caused by undesirable interactions occurring upon altered expression of the ID-containing protein.

Structure and intrinsic disorder in protein autoinhibition.

Autoinhibition plays a significant role in the regulation of many proteins. By analyzing autoinhibited proteins, we demonstrate that these proteins are enriched in intrinsic disorder because of the properties of their inhibitory modules (IMs). A comparison of autoinhibited proteins with structured and intrinsically disordered IMs revealed that in the latter group (1) multiple phosphorylation sites are highly abundant; (2) splice variants occur in greater number than in their structured cousins; and (3) activation is often associated with changes in secondary structure in the IM. Analyses of families of autoinhibited proteins revealed that the levels of disorder in IMs can vary significantly throughout homologous proteins, whereas residues located at the interfaces between the IMs and inhibited domains are conserved. Our findings suggest that intrinsically disordered IMs provide advantages over structured ones that are likely to be exploited in the fine-tuning of the equilibrium between active and inactive states of autoinhibited proteins.