Bulletin of the American Physical Society
APS March Meeting 2019
Volume 64, Number 2
Monday–Friday, March 4–8, 2019; Boston, Massachusetts
Session X64: Single Molecule Dynamics Inside and Outside of CellsFocus
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Sponsoring Units: DBIO Chair: Douglas Smith, Univ of California - San Diego Room: BCEC 259B |
Friday, March 8, 2019 8:00AM - 8:36AM |
X64.00001: Probing the dynamics of indiividual biomolecules with 1-μs resolution Invited Speaker: Thomas Perkins Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. To achieve state-of-the-art performance, we integrated several recent technical advances that improve the precision, stability, and accuracy of AFM-based single molecule force spectroscopy. Using modified cantilevers optimized for 1-µs resolution, we reexamined the unfolding of individual bacteriorhodopsin (bR) molecules in native lipid bilayers. The experimental data revealed the unfolding pathway in unprecedented detail. Numerous newly detected intermediates—many separated by as few as 2–3 amino acids—exhibited complex dynamics, including frequent refolding and state occupancies of <10 µs. Equilibrium measurements between such states enabled the folding free-energy landscape to be deduced. These results sharpen the picture of the mechanical unfolding of bR. Finally, recent efforts to improve the quantity and quality of AFM studies of diverse biomolecules, including nucleic-acid structures and globular proteins, will be discussed. |
Friday, March 8, 2019 8:36AM - 9:12AM |
X64.00002: Destabilization of nucleosomes by HMGB proteins and FACT complexes Invited Speaker: Mark Williams Nucleosome disruption plays a key role in many nuclear processes including transcription, DNA repair and recombination. We combine atomic force microscopy (AFM) and optical tweezers (OT) experiments to probe the effects of various motifs of High Mobility Group B (HMGB) proteins on nucleosome stability. We find that the double box Hmo1 and the single box Nhp6A from S. cerevisiae destabilize and unwind DNA from the H2A-H2B dimers that are part of the histone octamer. Unlike Nhp6A, Hmo1 also releases half of the DNA held by the (H3-H4)2 tetramer. Despite this destabilization, the octamers appear intact, and the remaining (H3-H4)2 tetramer interactions with the DNA are also destabilized. We also probe the human histone chaperone protein FACT (facilitates chromatin transcription), which also contains an HMGB box. We find that FACT (including the subunits Spt16 and SSRP1) binds directly to the nucleosome, specifically disrupting the H2A-H2B dimer interaction with DNA. Disruption leads to the release of that DNA from the nucleosome and the ejection of these dimers from the octamer, revealing a mechanism for regulation of chromatin assembly. Differences in nucleosome destabilization point to complementary roles that HMGB proteins play in chromatin remodeling. |
Friday, March 8, 2019 9:12AM - 9:24AM |
X64.00003: Single ligand and receptor binding spectroscopy using an atomic force microscopy Lina Alhalhooly, Matthew Confeld, Yongki Choi Binding and unbinding between individual ligands and receptors were measured and quantified using an atomic force microscopy (AFM). Initially, the cell stiffness is characterized by force-distance measurements using ligand-free AFM tips and compared to several cancer and non-cancerous cells. Then, we measured the unbinding force with peptide-coated AFM tips, targeting specific cell receptors. The unbinding force between the ligand and receptor was measured to be an order of 100 pN. Such individual, specific interaction measurements were further supported by pre-blocking the cell receptors with the excess, free ligands. |
Friday, March 8, 2019 9:24AM - 9:36AM |
X64.00004: Single Molecule Studies of a Novel Mechanism of Bacterial Transcription Initiation Debora Tenenbaum, Jane Kondev, Jeff Gelles In bacteria, gene transcription begins with the binding of an RNA polymerase (RNAP) core enzyme to the initiation factor σ, which allows it to recognize promoter sequences on the DNA and initiate RNA synthesis. In the canonical transcription cycle, upon reaching the termination sequence and releasing the transcript, RNAP detaches from the DNA and is free to restart the process. However, previous work in our lab has shown that following termination, bacterial RNAP frequently remains bound to the DNA template, and sometimes exhibits one-dimensional sliding over thousands of basepairs. Moreover, in the presence of free σ factor molecules in solution, the sliding, DNA-bound polymerase is often observed to restart transcription. This mechanism of transcription initiation might have implications for the transcriptional coupling of nearby genes. |
Friday, March 8, 2019 9:36AM - 9:48AM |
X64.00005: Single-molecule orientation mapping in 3D using a novel optical design Abhishek Kumar, James M Marr, Anthony Mautino, Mark McLean, Jeremiah Woodcock, Jefrey Gilman, Stephan Stranick, Veronika Szalai, James Liddle Single molecule (SM) localization-based super-resolution techniques have revolutionized the application of optical microscopy because of their ability to image structures below the diffraction limit in widefield. Fluorescent dyes and proteins used for SM imaging absorb and emit photons via an emission dipole, and any restriction on the motion of the dipole creates an asymmetry in the observation of the emission. Mapping this dipolar orientation can be a useful reporter of the local environment of molecules, and help improve localization accuracy for super-resolution imaging. We present a simple and flexible optical design, that can be incorporated into a standard optical microscope, to enable mapping the orientation of single molecules by simultaneously imaging 4 polarization orientations. A single camera is used to acquire all 4 channels, and fine adjustment controls for each channel minimize optical aberrations. We have used nanofabricated aperture arrays, fluorescent beads and single molecules embedded in a polymer matrix to characterize the system. |
Friday, March 8, 2019 9:48AM - 10:00AM |
X64.00006: Single molecule study of the stability of G-quadruplex DNA as an anti-cancer drug target Parastoo Maleki, Hamza Balci In targeted therapy, small molecule drugs are utilized to detect specific targets. G-quadruplex (GQ) structures are such a target for targeted cancer therapy. GQ is a physiologically significant secondary structure of DNA or RNA, which is formed by guanine-rich sequences. Studies on cancer cells show that stabilizing the GQ structures in telomeres with small molecules reduces telomerase activity and proliferation of cancer cells. However, the underlying dynamics of small molecule-GQ interactions are not known. In this study, we performed single molecule measurements to study how stability and dynamics of GQ are impacted by its interactions with several different small molecules. Moreover, we investigated the impact of these molecules on folding dynamics of the GQ structure. We observed significant enhancement in GQ stability in the presence of small molecules, as measured by the fraction of GQ molecules that remained folded against Bloom helicase mediated unfolding at the single molecule level. In addition, our measurements showed that folding of GQ takes place faster than our time resolution over all salt concentrations we studied (2-150 mM KCl) with or without small molecules, making it challenging to identify the impact of small molecules. |
Friday, March 8, 2019 10:00AM - 10:12AM |
X64.00007: Single-Molecule Activity of φ29 DNA Polymerase Monitored by Nanoscale Transistors Calvin Lau, Kristin Nichelle Gabriel, Narendra Kumar, Sudipta Majumdar, Arith J Rajapakse, Wonbae Lee, Gregory Weiss, Philip G Collins Single-molecule techniques are enabling the rapid advancement of next-generation DNA sequencing. Recently, electronic nanocircuits functionalized with DNA polymerases have arisen as a new, potentially high-density and high-throughput platform for single-molecule DNA sequencing and enzyme kinetic studies [1]. Here, we describe single-molecule electronic measurements of φ29 DNA polymerase, an enzyme having extremely high fidelity and processivity. Using φ29 linked to carbon nanotube transistors, base-by-base activity of individual polymerase molecules was continuously monitored for up to 30 min as the enzymes replicated single-stranded templates. Base incorporation rates averaged 20 s-1 but varied widely. Rates up to 200 and 400 s-1 occurred when φ29 encountered homopolymeric sequences of poly(dT) and poly(dC), respectively. On the other hand, the processing of poly(dA) and poly(dG) sequences was dominated by pauses lasting 50 to 300 s. Such observations of sequence-dependent activity demonstrate how single-molecule methods can excel over traditional, ensemble-based techniques like PCR sequencing and gel chromatography assays. |
Friday, March 8, 2019 10:12AM - 10:24AM |
X64.00008: Reliable extraction of energy landscape properties from critical force distributions Sudeep Adhikari, Kevin Beach The structural dynamics of biopolymers such as proteins are described in the context of their conformational energy landscapes. In pulling experiments using optical tweezers, features of the energy landscapes are extracted from the probability distribution of the critical force at which the polymer unfolds, and typically the analysis is based on rate equations having Bell-Evans form. We argue that this analysis is inadequate and leads to unreliable landscape parameters in many common situations. We propose a modified closed-form expression for the distribution of critical forces that works better for parameter extraction and is valid even up to fast pulling rates. We present results based on simulated data that confirm the utility of our new formula. |
Friday, March 8, 2019 10:24AM - 10:36AM |
X64.00009: Quantifying Collective Dynamics in the Ribosome Mariana Levi, Paul Whitford The functional dynamics of molecular assemblies often involve large-scale collective rearrangements. While these motions occur in a high-dimensional space, experiments are typically only able to simultaneously measure a small number of interatomic distances. Accordingly, a major challenge in single-molecule studies is to identify kinetically-relevant degrees of freedom. To address this issue for the ribosome, we use a range of theoretical models and molecular dynamics simulations in order to simulate hundreds of spontaneous large-scale (~30-50 Å) conformational transitions at various points of the elongation cycle. With these large data sets, we are assessing the ability of experimentally-accessible coordinates to capture the rate-limiting free-energy barriers. This analysis suggests design strategies for next-general experiments, as well as helps rationalize controversial/contradictory experimental observations. Finally, these calculations provide a quantitative foundation that is allowing us to study the precise relationship between structure and dynamics in the ribosome. |
Friday, March 8, 2019 10:36AM - 10:48AM |
X64.00010: Relaxation spectrum of a concentration quench of Brownian particles. Aykut Erbas, John Frederick Marko, Monica Olvera de la Cruz Single-molecule or SPR (Surface Plasmon Resonance) experiments rely on the relaxation of concentration quenches of initially surface-bound molecules into confined reservoirs to determine molecular kinetic rates. Similarly, biological processes such exocytosis, in which small molecules are emitted into the intracellular cleft for cellular communication, can be considered to be a relaxation process of an effective concentration quench. We study a model system closely related to the above cases in which weakly interacting Brownian particles are released from their binding sites into a confined volume by using molecular dynamics simulations and scaling arguments. Our results suggest that the rebinding rate of released particles exhibits various power laws until the confined volume is entirely filled by the particles. Furthermore, the cumulative rebinding rate, which is the time integration of the rebinding rate, exhibits a novel plateau behavior. This plateau is a result of the decreasing number of collisions between sparsely-placed binding sites and dissociated ligands. Our results can have important consequences for molecular signaling as well as for the interpretation of kinetic measurements of ligand-receptor interactions. |
Friday, March 8, 2019 10:48AM - 11:00AM |
X64.00011: Incorporating photo-artifacts into the analysis of single molecule FRET measurements Ioannis Sgouralis Single molecule experiments, such as those measuring FRET, encode precise information on individual molecules free of bulk averaging. Nevertheless, because smFRET assessments employ conventional fluorescence microscopy setups, artifacts such as background photons, shot-noise, cross-talk, and fluorophore blinking are limiting factors that necessitate the use of advanced analysis methods. In our recent work we employ infinite hidden Markov models and propose a novel formulation to model and analyze smFRET measurements that contains nonparametric statistics with an explicit representation of the fluorophore photo-physics. As a result, our method combines the advantages of traditional hidden Markov models while it avoids the associated state-space size restrictions or other model selection assumptions. For this, we carefully formulate the measurement process itself accounting for the photo-physics on the individual donors and acceptors in addition to the other experimental characteristics. As a result, our framework obtains accurate estimates even when recorded intensities are excessively noisy, photo-blinked, and also of short duration. Finally, our estimates remain accurate even when fluorophore photo-states alternate at scales comparable to those of the FRET efficiency itself. |
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