An efficient implicit-solvent model for self-assembled lipid bilayers is presented and analyzed using Langevin molecular dynamics simulations. The model is based on soft interactions between particles and short-range attractive interaction between lipid tails, leading for the self-assembly of a lipid bilayer without an explicit solvent. This allows for efficient simulations of large membranes over long times. The model exhibits a fluid phase at high temperatures and a gel phase at low temperatures, identified with the Lbeta-phase. The melting transition is investigated via analysis of the diffusivity of the lipid molecules, the chain-orientational order parameter, the sixfold bond-orientational order parameter, and the positional and bond-orientational correlation functions. The analysis suggests the existence of a hexatic phase over a narrow range of temperatures around the melting transition. The elastic properties of the membrane in the fluid phase are also investigated.
Blebs are balloon-shaped membrane protrusions that form during many physiological processes. Using computer simulation of a particle-based model for self-assembled lipid bilayers coupled to an elastic meshwork, we investigated the phase behavior and kinetics of blebbing. We found that blebs form for large values of the ratio between the areas of the bilayer and the cytoskeleton. We also found that blebbing can be induced when the cytoskeleton is subject to a localized ablation or a uniform compression. The results obtained are qualitatively in agreement with the experimental evidence and the model opens up the possibility to study the kinetics of bleb formation in detail.
The lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound.
The mechanism of helicase translocation on DNA remains controversial and the translocase activity driving their non-canonical functions such as protein displacement is poorly understood. Here, we used single molecule fluorescence assays to study a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, and discovered a progressive looping of ssDNA that is tightly coupled to PcrA translocation on DNA. Variance analysis of hundreds of looping events by a single protein demonstrated that PcrA translocates on ssDNA in uniform steps of 1 nt, reconciling discrepancies in previous structural and biochemical studies. On the forked DNA, rather than acting on the leading strand to unwind the duplex, PcrA anchored itself to the duplex junction and reeled in the lagging strand using its 3'-5' translocation activity. PcrA maintained the open conformation, not the closed conformation observed in crystallographic analysis, during looping-coupled translcation. This activity could rapidly dismantle a preformed RecA filament even at 1nM PcrA, suggesting that the translocation activity and structure-specific DNA binding are responsible for removal of potentially deleterious recombination intermediates.
to O1, O2, and O3 operators in the lac operon. Specifically, we demonstrated that LacI kept two supercoils within the 401 bp DNA-loop between O1 and O2 operators. Additionally, We carried out time course studies to determine the stability of the topological barriers that are produced by the different LacI-operator complexes. Our results showed that the stability of the topological barriers correlates with the DNA-binding affinity of LacI to the different operators i.e., O1, O2, O3, and Os operators. Furthermore, we confirmed our previous observation in which LacI is able to ''keep'' certain superhelical energy to stabilize LacI-lacO1 complexes. Our results can be explained by a model in which LacI behaves as a topological barrier in the lac operon to regulate the expression of lac-ZYA genes in Escherichia coli cells.
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