39 research outputs found
Single Molecule investigations of DNA Looping Using the Tethered Particle Method and Translocation by Acto-Myosin Using Polarized Total Internal Reflection Fluorescence Microscopy
Single molecule biophysics aims to understand biological processes by studying them at the single molecule level in real time. The proteins and nucleic acids under investigation typically exist in an aqueous environment within approximately ten degrees of room temperature. These seemingly benign conditions are actually quite chaotic at the nanoscale, where single bio-molecules perform their function. As a result, sensitive experiments and statistical analyses are required to separate the weak single molecule signal from its background. Protein-DNA interactions were investigated by monitoring DNA looping events in tethered particle experiments. A new analysis technique, called the Diffusive hidden Markov method, was developed to extract kinetic rate constants from experimental data without any filtering of the raw data; a common step that improves the signal to noise ratio, but at the expense of lower time resolution. In the second system, translocation of the molecular motor myosin along its actin filament track was studied using polarized total internal reflection (polTIRF) microscopy, a technique that determines the orientation and wobble of a single fluorophore attached to the bio-molecule of interest. The range of resolvable angles was increased 4-fold to include a hemisphere of possible orientations. As a result, the handedness of actin filament twirling as it translocated along a myosin-coated surface was determined to be left-handed. The maximum time resolution of a polTIRF setup was increased 50-fold, in part by recording the arrival times and polarization state of single photons using a modified time-correlated single photon counting device. A new analysis, the Multiple Intensity Change Point algorithm, was developed to detect changes in molecular orientation and wobble using the raw time-stamped data with no user-defined bins or thresholds. The analysis objectively identified changes in the orientation of a bifunctional-rhodamine labeled calmodulin that was attached to a myosin V molecule translocating along an actin filament. Long intervals corresponding to stable positions between tilting motions of the lever arm during each step were routinely observed. Substeps in the cycle that preceded these long dwells were measured, but only occasionally most likely because of the low number of photons detected during these rapid events
First-principles calculation of DNA looping in tethered particle experiments
We calculate the probability of DNA loop formation mediated by regulatory
proteins such as Lac repressor (LacI), using a mathematical model of DNA
elasticity. Our model is adapted to calculating quantities directly observable
in Tethered Particle Motion (TPM) experiments, and it accounts for all the
entropic forces present in such experiments. Our model has no free parameters;
it characterizes DNA elasticity using information obtained in other kinds of
experiments. [...] We show how to compute both the "looping J factor" (or
equivalently, the looping free energy) for various DNA construct geometries and
LacI concentrations, as well as the detailed probability density function of
bead excursions. We also show how to extract the same quantities from recent
experimental data on tethered particle motion, and then compare to our model's
predictions. [...] Our model successfully reproduces the detailed distributions
of bead excursion, including their surprising three-peak structure, without any
fit parameters and without invoking any alternative conformation of the LacI
tetramer. Indeed, the model qualitatively reproduces the observed dependence of
these distributions on tether length (e.g., phasing) and on LacI concentration
(titration). However, for short DNA loops (around 95 basepairs) the experiments
show more looping than is predicted by the harmonic-elasticity model, echoing
other recent experimental results. Because the experiments we study are done in
vitro, this anomalously high looping cannot be rationalized as resulting from
the presence of DNA-bending proteins or other cellular machinery. We also show
that it is unlikely to be the result of a hypothetical "open" conformation of
the LacI tetramer.Comment: See the supplement at
http://www.physics.upenn.edu/~pcn/Ms/TowlesEtalSuppl.pdf . This revised
version accepted for publication at Physical Biolog
Elementary simulation of tethered Brownian motion
We describe a simple numerical simulation, suitable for an undergraduate
project (or graduate problem set), of the Brownian motion of a particle in a
Hooke-law potential well. Understanding this physical situation is a practical
necessity in many experimental contexts, for instance in single molecule
biophysics; and its simulation helps the student to appreciate the dynamical
character of thermal equilibrium. We show that the simulation succeeds in
capturing behavior seen in experimental data on tethered particle motion.Comment: Submitted to American Journal of Physic
Changepoint Analysis for Single-Molecule Polarized Total Internal Reflection Fluorescence Microscopy Experiments
The experimental study of individual macromolecules has opened a door to determining the details of their mechanochemical operation. Motor enzymes such as the myosin family have been particularly attractive targets for such study, in part because some of them are highly processive and their “product” is spatial motion. But single-molecule resolution comes with its own costs and limitations. Often, the observations rest on single fluorescent dye molecules, which emit a limited number of photons before photobleaching and are subject to complex internal dynamics. Thus, it is important to develop methods that extract the maximum useful information from a finite set of detected photons. We have extended an experimental technique, multiple polarization illumination in total internal reflection fluorescence microscopy (polTIRF), to record the arrival time and polarization state of each individual detected photon. We also extended an analysis technique, previously applied to FRET experiments, that optimally determines times of changes in photon emission rates. Combining these improvements allows us to identify the structural dynamics of a molecular motor (myosin V) with unprecedented detail and temporal resolution
Concentration and Length Dependence of DNA Looping in Transcriptional Regulation
In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage), to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least) two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the J-factor for looping
Twirling of Actin by Myosins II and V Observed via Polarized TIRF in a Modified Gliding Assay
The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay in order to measure the rotation of an actin filament about its axis (“twirling”) as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeledactin monomers, using polarized total internal reflection (polTIRF) microscopy. In order to determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement four-fold. We found that whole myosin II and myosin V both twirl actin with a relatively long (~ µm), left-handed pitch that is insensitive to myosin concentration, filament length and filament velocity
Tethered Particle Motion as a Diagnostic of DNA Tether Length
The tethered particle motion (TPM) technique involves an analysis of the Brownian motion of a bead tethered to a slide by a single DNA molecule. We describe an improved experimental protocol with which to form the tethers, an algorithm for analyzing bead motion visualized using differential interference contrast microscopy, and a physical model with which we have successfully simulated such DNA tethers. Both experiment and theory show that the statistics of the bead motion are quite different from those of a free semiflexible polymer. Our experimental data for chain extension versus tether length fit our model over a range of tether lengths from 109 to 3477 base pairs, using a value for the DNA persistence length that is consistent with those obtained under similar solution conditions by other methods. Moreover, we present the first experimental determination of the full probability distribution function of bead displacements and find excellent agreement with our theoretical prediction. Our results show that TPM is a useful tool for monitoring large conformational changes such as DNA looping
Diffusive hidden Markov model characterization of DNA looping dynamics in tethered particle experiments
In many biochemical processes, proteins bound to DNA at distant sites are
brought into close proximity by loops in the underlying DNA. For example, the
function of some gene-regulatory proteins depends on such DNA looping
interactions. We present a new technique for characterizing the kinetics of
loop formation in vitro, as observed using the tethered particle method, and
apply it to experimental data on looping induced by lambda repressor. Our
method uses a modified (diffusive) hidden Markov analysis that directly
incorporates the Brownian motion of the observed tethered bead. We compare
looping lifetimes found with our method (which we find are consistent over a
range of sampling frequencies) to those obtained via the traditional
threshold-crossing analysis (which can vary depending on how the raw data are
filtered in the time domain). Our method does not involve any time filtering
and can detect sudden changes in looping behavior. For example, we show how our
method can identify transitions between long-lived, kinetically distinct states
that would otherwise be difficult to discern
Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay
The force generated between actin and myosin acts predominantly along the
direction of the actin filament, resulting in relative sliding of the thick and
thin filaments in muscle or transport of myosin cargos along actin tracks.
Previous studies have also detected lateral forces or torques that are
generated between actin and myosin, but the origin and biological role of these
sideways forces is not known. Here we adapt an actin gliding filament assay in
order to measure the rotation of an actin filament about its axis (twirling) as
it is translocated by myosin. We quantify the rotation by determining the
orientation of sparsely incorporated rhodamine-labeled actin monomers, using
polarized total internal reflection (polTIRF) microscopy. In order to determine
the handedness of the filament rotation, linear incident polarizations in
between the standard s- and p-polarizations were generated, decreasing the
ambiguity of our probe orientation measurement four-fold. We found that whole
myosin II and myosin V both twirl actin with a relatively long (micron),
left-handed pitch that is insensitive to myosin concentration, filament length
and filament velocity
DNA Looping Kinetics Analyzed Using Diffusive Hidden Markov Model
Tethered particle experiments use light microscopy to measure the position of
a micrometer-sized bead tethered to a microscope slide via a ~micrometer length
polymer, in order to infer the behavior of the invisible polymer. Currently,
this method is used to measure rate constants of DNA loop formation and
breakdown mediated by repressor protein that binds to the DNA. We report a new
technique for measuring these rates using a modified hidden Markov analysis
that directly incorporates the diffusive motion of the bead, which is an
inherent complication of tethered particle motion because it occurs on a time
scale between the sampling frequency and the looping time. We compare looping
lifetimes found with our method, which are consistent over a range of sampling
frequencies, to those obtained via the traditional threshold-crossing analysis,
which vary depending on how the raw data are filtered in the time domain. Our
method does not involve such filtering, and so can detect short-lived looping
events and sudden changes in looping behavior.Comment: 3 page pdf including 3 figures corrections: 2nd page, 1st column,
values of diffusion coefficient, spring constant and the decay time were
typed incorrectly. No conlcusions were affecte