Simulated single molecule microscopy with SMeagol

SMeagol is a software tool to simulate highly realistic microscopy data based on spatial systems biology models, in order to facilitate development, validation, and optimization of advanced analysis methods for live cell single molecule microscopy data. Availability and Implementation: SMeagol runs on Matlab R2014 and later, and uses compiled binaries in C for reaction-diffusion simulations. Documentation, source code, and binaries for recent versions of Mac OS, Windows, and Ubuntu Linux can be downloaded from http://smeagol.sourceforge.net.Comment: v2: 14 pages including supplementary text. Pre-copyedited, author-produced version of an application note published in Bioinformatics following peer review. The version of record, and additional supplementary material is available online at: https://academic.oup.com/bioinformatics/article-lookup/doi/10.1093/bioinformatics/btw10

Force generation in small ensembles of Brownian motors

The motility of certain gram-negative bacteria is mediated by retraction of type IV pili surface filaments, which are essential for infectivity. The retraction is powered by a strong molecular motor protein, PilT, producing very high forces that can exceed 150 pN. The molecular details of the motor mechanism are still largely unknown, while other features have been identified, such as the ring-shaped protein structure of the PilT motor. The surprisingly high forces generated by the PilT system motivate a model investigation of the generation of large forces in molecular motors. We propose a simple model, involving a small ensemble of motor subunits interacting through the deformations on a circular backbone with finite stiffness. The model describes the motor subunits in terms of diffusing particles in an asymmetric, time-dependent binding potential (flashing ratchet potential), roughly corresponding to the ATP hydrolysis cycle. We compute force-velocity relations in a subset of the parameter space and explore how the maximum force (stall force) is determined by stiffness, binding strength, ensemble size, and degree of asymmetry. We identify two qualitatively different regimes of operation depending on the relation between ensemble size and asymmetry. In the transition between these two regimes, the stall force depends nonlinearly on the number of motor subunits. Compared to its constituents without interactions, we find higher efficiency and qualitatively different force-velocity relations. The model captures several of the qualitative features obtained in experiments on pilus retraction forces, such as roughly constant velocity at low applied forces and insensitivity in the stall force to changes in the ATP concentration.Comment: RevTex 9 pages, 4 figures. Revised version, new subsections in Sec. III, removed typo

Anisotropic membrane curvature sensing by amphipathic peptides

Many proteins and peptides have an intrinsic capacity to sense and induce membrane curvature, and play crucial roles for organizing and remodelling cell membranes. However, the molecular driving forces behind these processes are not well understood. Here, we describe a new approach to study curvature sensing, by simulating the direction-dependent interactions of single molecules with a buckled lipid bilayer. We analyse three amphipathic antimicrobial peptides, a class of membrane-associated molecules that specifically target and destabilize bacterial membranes, and find qualitatively different sensing characteristics that would be difficult to resolve with other methods. These findings provide new insights into the curvature sensing mechanisms of amphipathic peptides and challenge existing theories of hydrophobic insertion. Our approach is generally applicable to a wide range of curvature sensing molecules, and our results provide strong motivation to develop new experimental methods to track position and orientation of membrane proteins.Comment: 14 pages. Supplementary movies available on request. Accepted version, with corrected description lipid bilayer compositio

Gas phase thermometry of hot turbulent jets using laser induced phosphorescence

This article is made available through the Brunel Open Access Publishing Fund. Copyright @ 2013 OSAThe temperature distributions of heated turbulent jets of air were determined using two dimensional (planar) laser induced phosphorescence. The jets were heated to specific temperature increments, ranging from 300 – 850 K and several Reynolds numbers were investigated at each temperature. The spectral ratio technique was used in conjunction with thermographic phosphors BAM and YAG:Dy, individually. Single shot and time averaged results are presented as two dimensional stacked images of turbulent jets. YAG:Dy did not produce a high enough signal for single shot measurements. The results allowed for a direct comparison between BAM and YAG:Dy, revealing that BAM is more suitable for relatively lower temperature, fast and turbulent regimes and that YAG:Dy is more suited to relatively higher temperature, steady flow situations

Decay times in turnover statistics of single enzymes

The first passage times for enzymatic turnovers in non-equilibrium steady state display a statistical symmetry property related to non-equilibrium fluctuation theorems, that makes it possible to extract the chemical driving force from single molecule trajectories in non-equilibrium steady state. Below, we show that this system violates the general expectation that the number of decay constants needed to fit a first passage time distribution reflects the number of states in the escape problem. In fact, the structure of the kinetic mechanism makes half of the decay times vanish identically from the turnover time distribution. The terms that cancel out correspond to the eigenvalues of a certain sub-matrix of the master equation matrix for the first exit time problem. We discuss how these results make modeling and data analysis easier for such systems, and how the turnovers can be measured.Comment: 4 pages, 1 figure v2: Published version, minor corrections in response to referee comment

Multiple Lac-mediated loops revealed by Bayesian statistics and tethered particle motion

The bacterial transcription factor LacI loops DNA by binding to two separate locations on the DNA simultaneously. Despite being one of the best-studied model systems for transcriptional regulation, the number and conformations of loop structures accessible to LacI remain unclear, though the importance of multiple co-existing loops has been implicated in interactions between LacI and other cellular regulators of gene expression. To probe this issue, we have developed a new analysis method for tethered particle motion, a versatile and commonly-used in vitro single-molecule technique. Our method, vbTPM, performs variational Bayesian inference in hidden Markov models. It learns the number of distinct states (i.e., DNA-protein conformations) directly from tethered particle motion data with better resolution than existing methods, while easily correcting for common experimental artifacts. Studying short (roughly 100 bp) LacI-mediated loops, we provide evidence for three distinct loop structures, more than previously reported in single-molecule studies. Moreover, our results confirm that changes in LacI conformation and DNA binding topology both contribute to the repertoire of LacI-mediated loops formed in vitro, and provide qualitatively new input for models of looping and transcriptional regulation. We expect vbTPM to be broadly useful for probing complex protein-nucleic acid interactions.Comment: 34 pages, 25 figures, including Supporting information. To appear in Nucleic Acids Research. Accompanying open-source software: http://sourceforge.net/projects/vbtpm