Browsing by Author "Harris, Nolan C."

Now showing 1 - 4 of 4
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    Analyzing Single-Molecule Manipulation Experiments
    (2008-10) Calderon, Christopher P.; Harris, Nolan C.; Kiang, Ching-Hwa; Cox, Dennis D.
    Single-molecule manipulation studies can provide quantitative information about the physical properties of complex biological molecules without ensemble artifacts obscuring the measurements. We demonstrate computational techniques which aim at more fully utilizing the wealth of information contained in noisy experimental time series. The "noise" comes from multiple sources, e.g. inherent thermal motion, instrument measurement error, etc. The primary focus of this article is a methodology for using time domain based methods for extracting the effective molecular friction from single-molecule pulling data. We studied molecules composed of 8 tandem repeat titin I27 domains, but the modeling approaches have applicability to other single-molecule mechanical studies. The merits and challenges associated with applying such a computational approach to existing single-molecule manipulation data are also discussed.
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    Nanoscale manipulation and studies of individual biomolecules and DNA-based nanostructures
    (2009) Harris, Nolan C.; Kiang, Ching-Hwa
    Nanoscale manipulation of individual biomolecules, using such techniques as the atomic force microscope (AFM) and laser optical tweezers (LOT), has increased the scope and detail with which important biological interactions, such as protein folding, receptor-ligand binding, and double-stranded DNA melting, can be studied. In recent years, single molecule manipulation via AFM has been used to characterize the mechanical properties of various proteins. However, since single molecule manipulation experiments are typically performed under nonequilibrium conditions, extracting thermodynamic properties from these measurements has proven difficult. The derivation of Jarzynski's equality, which relates nonequilibrium work fluctuations to equilibrium free energy differences, provides the possibility for extracting equilibrium information from single molecule manipulation data. Here, single molecule force measurements of the stretching and unfolding of the titin I27 protein are analyzed using Jarzynski's equality to reconstruct the underlying free energy landscape associated with this process. We describe the procedures for the automated selection, alignment, and Jarzynski analysis of single molecule data. We use the recovered equilibrium free energy landscape to estimate thermodynamic properties such as the unfolding free energy barrier, which is in good agreement with estimates from bulk kinetics studies and various simulations. Also, the convergence behavior of Jarzynski's equality with respect to pulling velocity is studied experimentally. We demonstrate that with enough pulling trajectories, Jarzynski's equality will indeed recover the equilibrium free energy landscape for a given process. The number of trajectories required to recover the equilibrium free energy for a given pulling velocity is used to quantify this convergence behavior and identify a range of pulling velocities that is most efficient for thermodynamic analysis of single molecule manipulation data. Single molecule manipulation is also used to characterize DNA melting transitions by repeatedly stretching and relaxing an individual λ-DNA molecule. Here, a force induced transition between B form DNA ( B -DNA) and S form DNA ( S -DNA), prior to dsDNA melting, is observed. The mechanical properties of the various conformations, B -DNA, S -DNA, and single-stranded DNA, are quantified using polymer elasticity models, and are shown to agree well with expectations from previous experiments and theory. Fabrication of DNA-based nanostructures, particularly DNA-gold nanoparticle assemblies, has generated significant interest due to their interesting optical and phase transition properties. Here, the effects of various types of disorder within the DNA-gold nanoparticle system are studied and quantified. We show that the stability of these complex fluids can not be quantified using expectations from free DNA hybridization. For example, when linking pairs of gold nanoparticle probes using a DNA linker sequence, a lack of base-pairing symmetry between the probes creates a disorder that decreases the overall stability of the system. This occurs despite the energy contribution that is gained by adding a single base pair to one probe, creating the lack of symmetry. The assumption that base-pairing defects will lower the stability of the nanoparticle aggregates due a loss of hybridization energy is also found to be violated with surprising frequency. In some cases, nonspecific binding between the gold particle surface and a free DNA base can result in a system with increased stability, despite the loss in energy resulting from a mismatched or deleted base. These observations demonstrate that the DNA interactions within these nanostructures are highly complex and that the system stability is not always governed by free DNA hybridization.
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    Quantifying DNA Melting Transitions Using Single-Molecule Force Spectroscopy
    (2008-09) Calderon, Christopher P.; Chen, Wei-Hung; Lin, Kuan-Jiuh; Harris, Nolan C.; Kiang, Ching-Hwa
    We stretched a DNA molecule using atomic force microscope and quantified the mechanical properties associated withﾠBandﾠSﾠforms of double-stranded DNA (dsDNA), molten DNA, and single-stranded DNA (ssDNA). We also fit overdamped diffusion models to the AFM time series and used these models to extract additional kinetic information about the system. Our analysis provides additional evidence supporting the view that S-DNA is a stable intermediate encountered during dsDNA melting by mechanical force. In addition, we demonstrated that the estimated diffusion models can detect dynamical signatures of conformational degrees of freedom not directly observed in experiments.
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    Quantifying Multiscale Noise Sources in Single-Molecule Time Series
    (2008-09) Calderon, Christopher P.; Harris, Nolan C.; Kiang, Ching-Hwa; Cox, Dennis D.
    When analyzing single-molecule data, a low-dimensional set of system observables typically serve as the observational data. We calibrate stochastic dynamical models from time series that record such observables. Numerical techniques for quantifying noise from multiple time-scales in a single trajectory, including experimental instrument and inherent thermal noise, are demonstrated. The techniques are applied to study time series coming from both simulations and experiments associated with the nonequilibrium mechanical unfolding of titin's I27 domain. The estimated models can be used for several purposes: (1) detect dynamical signatures of "rare events" by analyzing the effective diffusion and force as a function of the monitored observable, (2) quantify the influence that conformational degrees of freedom, which are typically difficult to directly monitor experimentally, have on the dynamics of the monitored observable, (3) quantitatively compare the inherent thermal noise to other noise sources, e.g. instrument noise, variation induced by conformational heterogeneity, etc., (4) simulate random quantities associated with repeated experiments, (5) apply pathwise, i.e. trajectory-wise, hypothesis tests to assess the goodness-of-fit of the models and even detect conformational transitions in noisy signals. These items are all illustrated with several examples.