Search

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 spectral and photophysical characteristics of the autofluorescent proteins were analyzed and compared to flavinoids to test their applicability for single-molecule microscopy in live cells. We compare 1) the number of photons emitted by individual autofluorescent proteins in artificial and in vivo situations, 2) the saturation intensities of the various autofluorescent proteins, and 3) the maximal emitted photons from individual fluorophores in order to specify their use for repetitive imaging and dynamical analysis. It is found that under relevant conditions and for millisecond integration periods, the autofluorescent proteins have photon emission rates of approximately 3000 photons/ms (with the exception of DsRed), saturation intensities from 6 to 50 kW/cm2, and photobleaching yields from 10(-4) to 10(-5). Definition of a detection ratio led to the conclusion that the yellow-fluorescent protein mutant eYFP is superior compared to all the fluorescent proteins for single-molecule studies in vivo. This finding was subsequently used for demonstration of the applicability of eYFP in biophysical research. From tracking the lateral and rotational diffusion of eYFP in artificial material, and when bound to membranes of live cells, eYFP is found to dynamically track the entity to which it is anchored.

Essentially all approaches to artificial molecular motors rely on diffusional stepping, i.e. the free energy input that powers the motor is used to rectify thermal motion. However, many models for biological motors, e.g. myosins or kinesins, include directed motion due to a “power stroke”. In this project we are employing a bottom-up approach to developing a relatively simple experimental model system, the “Inchworm”, with the intention to create the first artificial motor with a power stroke. Specifically, the Inchworm consists of a stretch of DNA confined inside a nanochannel, with ligand-gated repressor proteins immobilized on the nanochannel walls. By cyclically stretching and contracting the Inchworm DNA, via changes in buffer ionic strength, and simultaneously externally controlling the DNA repressor proteins’ binding and unbinding states, the motor is designed to achieve processive and unidirectional motion along the nanochannel. Here we report the status of this project: we have constructed the Inchworm DNA, expressed the ligand-gated repressor proteins and tested their specificity. In addition, we have developed a protocol for immobilization of the repressor proteins to the nanochannel walls and produced nanochannels (100-200 nm) in quartz, which can be interfaced with preexisting micro-fluidic systems.Acknowledgment: This work was supported by MONAD, HFSP, the Swedish Research Council and nmC@LU.

Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom-up approach to understanding biological motors and present a class of designs for synthetic protein motors that move along a linear DNA track.This presentation will focus on two aspects of the motor design. Firstly the use of self-assembling components whose function is to co-localise the necessary motor functions. We demonstrate that a coiled coil template can be adapted in order to program the self-assembly of three different coiled coils from a solution of six peptides.Secondly the role of linkers between components in controlling dynamics and hence both processivity and power strokes within our motor designs will be discussed. The use of cis-trans isomerization of azobenzene as a mechanism for driving conformational change within the motor will be demonstrated.1. The Tumbleweed: towards a synthetic protein motor, EHC Bromley et al. HFSP J. 3, 204-212 (2009).2. Tuning the performance of an artificial protein motor, NJ Kuwada et al. Phys. Rev. E 84(3) 031922 (2011).3. Designed α-Helical Tectons for Constructing Multicomponent Synthetic Biological Systems EHC Bromley et al. J. Am. Chem. Soc. 131, 928-930 (2009).4. Squaring the circle in peptide assembly: From fibers to discrete Nanostructures by de Novo Design. AL Boyle et al. J. Am. Chem. Soc. 134, 15457-15467 (2012).

Optical tweezers have become powerful tools to manipulate biomolecular systems, but are increasingly difficult to use when the size of the molecules is

International audience; We present a novel readout scheme for gold nanoparticle-based DNA microarrays relying on Laser Induced Scattering around a NanoAbsorber (LISNA). It provides direct counting of individual nanoparticles present on each array spot and stable signals, without any silver enhancement. Given the detection of nanometer-sized particles, which minimize the steric hindrance, the linear dynamic range of the method is particularly large and well suited for microarray detection.

Molecular spiders are synthetic molecular motors featuring multiple legs such that each leg can interact with a substrate through binding and cleavage. Experimental studies of molecular spiders suggest that the motion of the spider on both a substrate matrix [R. Pei et al., J. Amer. Chem. Soc.128, 12693 (2006)] and a two dimensional substrate track [K. Lund et al, Nature 465, 206 (2010)] is biased towards uncleaved substrates. We first investigated the origin of the spider's biased motion by using Monte Carlo simulations of bipedal spiders on a 1D track based on a realistic chemical kinetic model [L. Samii et al., Phys. Rev. E81, 021106-1 (2010)], and found that substrate cleavage and spider detachment from the track both contribute to biased motion of the spider population. In the work reported here, we extend these studies by investigating how experimental parameters such as number of legs, leg length and substrate cleavage rate can be tuned to optimize the motor properties of the spider. We find that each of these parameters affect properties such as binding time to the track, processivity and speed in different ways. To evaluate further the motor properties of the spider, we calculated the thermodynamic efficiencies of multi-pedal molecular spiders, and found that they are both force and time dependent. By comparing our results with biological molecular motors such as kinesin, we were able to comment on the effect of tight versus loose mechano-chemical coupling on thermodynamic efficiency.

Nonlinear microscopy, which in combination with fluorescence spectroscopy is called nonlinear spectral imaging (NLSI), affords access to specimen morphology and (auto)fluorescence spectra at the same time. Thus, it measures the biochemical composition, while also distinguishing different parts of the tissue. Data interpretation is simplified by a new analysis method, the spectral phasor [1], which results in a robust, quick, and semi-blind spectral unmixing of fluorescent species.NLSI and the spectral phasor are a very user-friendly technique and have the potential to address a broad range of microbiological questions. We introduce them as a novel minimum-invasive technique to monitor the state of "fungal cells" (hyphae). Fungi, both used as consumables and organisms to produce industrial and pharmaceutical compounds, require stringent quality control during their growth. To this end, and as one possible application of NLSI and the spectral phasor, we present their use in monitoring the quality and freshness of white button mushrooms.[1] F. Fereidouni, A.N. Bader, and H.C. Gerritsen, Optics Express, 20,12729-12741 (2012).RGB-representation of a nonlinear spectral image of the white button mushroom (A. bisporus, left) and the corresponding phasor.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Based on a recent approach to understanding protein-based molecular motors (1), we present computer simulations of a novel nanomotor concept dubbed the Synthetic Kinesin Analog Motor Protein (SKAMP). SKAMP is a purely diffusive linear motor consisting of four ligand gated DNA-binding (repressor) proteins of two types, A and B, linked by three rigid coiled-coils (‘rods’) to form a complex A1-A2-B2-B1, the length of the central rod being shorter than the outer two. Directional stepping along a periodic DNA track is maintained by a temporally periodic external chemical supply. Due to the shorter central rod, SKAMP makes use of a mechanism analogous to that used by kinesin which involves docking of a neck linker onto a motor domain. This mechanism allows kinesin to reduce its diffusional search time for a binding site. We use coarse-grained Langevin Dynamics simulations in the overdamped limit to study the detailed motion of SKAMP when subject to a load force, SKAMP acting as a shuttle and the increase of SKAMP's performance by external feedback.1. Bromley et al. HFSP Journal 3 204-213 (2009).View Large Image | View Hi-Res Image | Download PowerPoint Slide

In a bottom up approach to understanding molecular motors, a synthetic protein-based molecular motor, the “tumbleweed”, is being designed and constructed [1]. This design uses three ligand dependent DNA repressor proteins to rectify diffusive motion of the construct along a DNA track. These proteins are MetJQ44K, TrpR, and DtxR. To predict the behavior of this artificial motor one needs to understand the binding and unbinding kinetics of the repressor proteins at a single-molecule level. An assay, similar to tethered particle motions assays [2], is used to measure the unbinding rates of these three DNA repressor proteins. In this assay the repressor is immobilized to a surface in a microchamber. Long DNA with the correct recognition sequence for one of the repressors is attached to a streptavidin-coated microsphere. As the DNA-microsphere construct diffuses through the microchamber it will sometimes bind to the repressor protein. Using brightfield microscopy and a CCD camera the diffusive motion of the microsphere can be characterized and bound and unbound states can be differentiated. On the order of ten microspheres can be easily visualized at one time allowing single-molecule measurements to be done in parallel. The resulting kinetic measurements are compared to bulk binding kinetics measured in a QCM device.[1] EHC Bromley et al. 2009. The Tumbleweed: Towards a synthetic protein motor. HFSP Journal. 3:204-212.[2] F Vanzi et al. 2006. Lac Repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion. Nuc. Acids. Res. 34:3409-3420.

Protein-mediated DNA looping is a ubiquitous motif in transcriptional control schemes. Formation of these loops is driven by thermal fluctuations of the substrate DNA, which in turn are known to be exquisitely sensitive to mechanical constraints on the DNA. Because DNA in vivo is subject to a complex micromechanical environment, it is intriguing to study the effect of mechanical tension in the substrate DNA on the formation of these regulatory loops to investigate the role of mechanics in controlling gene regulation. For this purpose, we measured the formation and breakdown rates of lac repressor-mediated DNA loops under tension using constant-force axial optical tweezers.We observed that an incremental force of less than 100 femtonewtons is sufficient to reduce loop formation rate about sevenfold in a construct with an inter-operator spacing of 305 bp. This result suggests the possibility of mechanical pathways to control gene expression with forces that are two orders of magnitude lower than other typical intracellular forces acting on DNA, such as the forces exerted by RNA polymerase and molecular motors. Moreover, we developed a model that quantifies the relation between the force sensitivity of the loop formation rate and the angle between the incoming and outgoing DNA strand in the loop as a way to infer loop topology from our micromechanical measurements. We conclude that the LacI-mediated DNA loop prefers an anti-parallel loop topology over a parallel conformation.

International audience; In the last decade evidence has accumulated that small domains of 50-700 nm in diameter are located in the exoplasmic leaflet of the plasma membrane. Most of these domains supposedly consist of specific sets of lipids and proteins, and are believed to coordinate signal transduction cascades. Whether similar domains are also present in the cytoplasmic leaflet of the plasma membrane is unclear so far. To investigate the presence of cytoplasmic leaflet domains, the H-Ras membrane-targeting sequence was fused to the C-terminus of the enhanced yellow fluorescent protein. Using single-molecule fluorescence microscopy, trajectories of individual molecules diffusing in the cytoplasmic leaflet of the plasma membrane were recorded. From these trajectories, the diffusion of individual membrane-anchored enhanced yellow fluorescent protein molecules was studied in live cells on timescales from 5 to 200 ms. The results show that the diffusion of 30-40% of the molecules is constrained in domains with a typical size of 200 nm. Neither breakdown of actin nor cholesterol extraction changed the domain characteristics significantly, indicating that the observed domains may not be related to the membrane domains identified so far.

L-type Ca 2+ channels are an important means by which a cell regulates the Ca 2+ influx into the cytosol on electrical stimulation. Their structure and dynamics in the plasma membrane, including their molecular mobility and aggregation, is of key interest for the in-depth understanding of their function. Construction of a fluorescent variant by fusion of the yellow-fluorescent protein to the ion channel and expression in a human cell line allowed us to address its dynamic embedding in the membrane at the level of individual channels in vivo. We report on the observation of individual fluorescence-labeled human cardiac L-type Ca 2+ channels using wide-field fluorescence microscopy in living cells. Our fluorescence and electrophysiological data indicate that L-type Ca 2+ channels tend to form larger aggregates which are mobile in the plasma membrane.

Though the biological function of many natural molecular motors is fairly well established, many structure-function details responsible for motor performance remain vague or unknown completely. Recently, we have undertaken a new bottom-up approach to understanding biological molecular motors by designing and building an artificial, protein-based molecular motor dubbed the Tumbleweed (TW). The TW is a purely diffusive motor construct consisting of three DNA-binding proteins attached to a designed, protein-based central hub, where directional stepping along a DNA track is maintained by a temporally periodic external chemical supply. To better understand important design and performance characteristics of the TW, coarse-grained Langevin Dynamics (LD) simulations and numerical solutions to the Master Equation (ME) were carried out. The LD approach, which is a single motor simulation, is particularly suitable for exploring the diffusional behavior of the system, where the ME approach, which models an ensemble of motor states, is best suited for statistically exploring the parameter space of the system and the interaction of processes at different time scales. We present results from these two theoretical approaches that illuminate not only important design and experimental considerations, such as motor geometry and track spacing, but also produce unexpected diffusional behavior. Of particular interest is that the addition of certain internal symmetric potentials can increase motor performance. For example, the addition of a non-specific binding potential, symmetric about the DNA track, can double motor speed by replacing some of the 3D diffusional search by a relatively fast 1D diffusional slide along the DNA. This, and other symmetric potential inputs that increase motor performance by subtly amplifying asymmetries in the system, are not only fundamentally interesting but also may be applicable to any molecular motor that incorporates a diffusional search in its stepping cycle.

Molecular spiders are synthetic molecular motors featuring multiple legs that each can interact with a substrate through binding and cleavage. Experimental studies suggest the motion of the spider in a matrix is biased towards uncleaved substrates, and that spider properties such as processivity can be altered by changing the binding strength of the legs to substrate [R. Pei et al., J. Amer. Chem. Soc.128, 12693 (2006)]. We investigate the origin of biased motion and molecular motor properties of bipedal spiders using Monte Carlo simulations. Our simulations combine a realistic chemical kinetic model, hand-over-hand (HOH) or inchworm (IW) modes of stepping, and the use of a 1D track. We find that substrate cleavage and spider detachment from the track are both contributing mechanisms to population bias but are not necessary for biased motion on an asymmetric track. We investigate the contributions of stepping mechanism to speed, randomness parameter, processivity, coupling and efficiency, and comment on how these molecular motor properties can be altered by changing experimentally tunable kinetic parameters. We then consider the more general case where steps can occur by any mechanism, subject to steric constraints. We compare these results with the above for bipedal spiders and then simulate quadrupedal spiders to investigate the effect of leg number on motor performance.