Engineering ClpXP for Single-Molecule Protein Sequencing

Sunday, February 12, 2017 tracked with changes in diffusion time of the larger mAb-antigen complex. With this approach, we measured the binding affinities of Alexa-488 labeled streptavidin and anti-streptavidin immunoglobulin G2 (IgG2) in buffer and neat serum, and found that binding is ~3-fold tighter in serum versus buffer. Through control experiments in viscosity-matched sucrose solution, we found that differences in viscosity may account for some but not all of this effect. Serum contains high concentrations of co-solutes of various sizes including albumin and g-globulins. We tested the effects of macromolecular crowding on binding using physiological concentrations of bovine serum albumin as well as polymeric crowders, and found that crowding also partially accounts for the tighter binding of the streptavidin-IgG2 pair in serum. Conversely, serum does not affect the binding affinity of anti-streptavidin IgG1 to streptavidin. Accurate characterization of therapeutic proteins in relevant conditions is integral to assessing their safety and efficacy. The implications of these results with respect to drug development are discussed. 739-Pos Board B504 In Vitro Binding of 6S RNA Mango to RNA Polymerase by Two Photon Fluorescence Cross Correlation Spectroscopy S. Shyam Sundar Panchapakesan1, Eric J. Hayden2, Peter Unrau1, Matthew L. Ferguson3. 1 Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada, 2Department of Biological Science, Boise State University, Boise, ID, USA, 3Department of Physics, Boise State University, Boise, ID, USA. The assembly of RNA and protein complexes is a fundamental process for life but has been difficult to study both in vitro and in vivo. Recently a novel aptamer based method to fluorescently label RNA molecules has been reported(Dolgosheina et al. 2014). Here we report the successful utilization of the RNA Mango þ thiazole orange derivative to measure the in vitro binding of 6S RNA to RNA Polymerase from E. coli. By two photon Fluorescence Cross Correlation Spectroscopy, we are able to independently measure the molecular brightness, diffusion coefficient and concentration of 6S RNA Mango, RNA Polymerase GFP and complex as we increase RNA Polymerase concentration giving us an estimate for the equilibrium binding constant of the complex in vitro. This experiment demonstrates the utility of RNA Mango for both in vitro and in vivo single molecule experiments. Dolgosheina, Elena V., Sunny C. Y. Jeng, Shanker Shyam S. Panchapakesan, Razvan Cojocaru, Patrick S. K. Chen, Peter D. Wilson, Nancy Hawkins, Paul A. Wiggins, and Peter J. Unrau. 2014. ‘‘RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking.’’ ACS Chemical Biology 9 (10): 2412-20. 740-Pos Board B505 Single-Molecule Fluorescence Study of RNA Recognition by Viral RNAi Suppressors Mohamed Fareh1, Jasper van Lopik1, Iason Katechis1, Ronald van Rij2, Chirlmin Joo1. 1 Kavli Institute of Nanoscience, Bionanoscience Department, Delft University of Technology, Delft, Netherlands, 2Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands. RNA interference (RNAi) is an indispensable antiviral defense mechanism in insects including mosquitoes that transmit human diseases such as dengue or Zika fever. To escape the cellular defense, viruses employ various proteins called viral suppressors of RNAi (VSRs). VSRs are thought to suppress the RNAi pathway at several different levels. This interaction precludes the recognition and elimination of viral RNA, leading to viral survival and proliferation in detriment of the host organism. Despite a decade of research on VSRs, how VSRs antagonize RNAi remains incompletely understood. Here we employed single-molecule fluorescence techniques to investigate how VSRs inhibit viral RNA recognition by Dicer-2, a key enzyme in RNAi antiviral defense. Our single-molecule data showed that the majority of VSRs tested directly interact with double-stranded RNA (dsRNA), a viral replication intermediate. The stable binding inhibits recognition of viral dsRNA by Dicer-2 and consequently suppresses the RNAi antiviral pathway. The length of the double-stranded stem region is a crucial feature in forming a stable interaction between viral dsRNA and VSRs. 741-Pos Board B506 Dual Role of Munc13 in Regulating SNARE Assembly for Fast Neurotransmitter Release Ucheor Brandon Choi. Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.

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Neuronal SNAREs (synaptobrevin, syntaxin, SNAP-25) are the minimal fusion machinery, but require synaptotagmin, complexin, Munc13, and Munc18 for fast Ca2þ-triggered neurotransmitter release. Munc13s have a central role in synaptic vesicle priming through their MUN domain. Using single molecule FRET we identified two distinct roles of the MUN domain in SNARE complex assembly for efficient Ca2þ-triggered fusion events. Prior to SNARE complex assembly syntaxin adopts a closed conformation tightly bound to Munc18. We found that the MUN domain changes the conformation of the linker region of syntaxin when bound to Munc18 providing a nucleation site for ternary SNARE complex formation. Interestingly we found an additional role of the MUN domain where it cooperates with Munc18 to ensure properly assembled SNARE complex, which resulted in a dramatic increase in Ca2þ-triggered fusion efficiency and Ca2þ-sensitivity. 742-Pos Board B507 Monitoring Small Molecule and G-Quadruplex Interactions and Kinetics using Single Molecule FRET Parastoo Maleki. Kent State University, Kent, OH, USA. Telomestatin and oxazole telomestatin derivatives (OTD) are small molecules stabilizing G-quadruplex (GQ) structures and are prominent due to their anticancer drug potential. Despite the observation of enhanced thermodynamic stability imparted by such small molecules on GQ, the underlying dynamics of small molecule-GQ interactions are not known. To have a better understanding of these interactions we employed single molecule Fo¨rster resonance energy transfer (smFRET) to study the system, where we utilized a Cy5-labeled OTD (L1Cy5-7OTD). These studies demonstrate that interactions of this small molecule with GQ are dynamic in terms of binding kinetics and possibly in terms of rotational freedom. The Cy5 fluorophore has enabled monitoring and quantifying binding, dwell, and dissociation of a single L1Cy5-7OTD molecule as it interacted with GQ, which to our knowledge has not been demonstrated for any GQ stabilizing small molecule before. We show that L1Cy5-7OTD remains bound to GQ for tens of seconds, with significantly longer dwell times and higher binding frequencies for more stable GQ. In addition, we propose L1Cy5-7OTD to have at least two preferred primary binding orientations and is able to transition between these orientations while it remains stacked on a G-tetrad. 743-Pos Board B508 Engineering ClpXP for Single-Molecule Protein Sequencing Mike Filius, Jetty van Ginkel, Chirlmin Joo. BioNanoScience, Delft University of Technology, Delft, Netherlands. Proteins are vital in all biological systems as they are involved in a large number of structural and functional pathways. Mass sprectometric techniques have been proposed to provide valuable tools for complete proteomic studies of the human proteome. However, current mass spectrometers lack the sensitivity for determining the least abundant proteins within a cell. A single-molecule method that analyses the proteins molecule by molecule may have the sensitivity to shed light of the least abundant proteins within a single cell. The major challenge of protein sequencing lies in the fact that protein sequences consist of 20 different amino acids. Here, we aim to develop a novel protein sequencing technique that could identify proteins based on their fingerprint at the single-molecule level. In the novel method, proteins will be identified based on two types of amino acids only instead of all 20 types. In the present study we demonstrate since-molecule FRET detection of an acceptor labeled protein substrate by a donor labeled protein analyzer ClpXP. This highly sensitive approach holds promise to detect the least abundant proteins of a cell, that can have high value for biomedical sciences. 744-Pos Board B509 Investigating the Mechanism of Ultra-Fast Energy Transfer between Venus Oligomers using Time-Resolved Anisotropy, Fluorescence Correlation Spectroscopy, and Photon Antibunching Youngchan Kim1, Grace H. Taumoefolau1, Tuan A. Nguyen1, Henry L. Puhl1, Paul S. Blank2, Steven S. Vogel1. 1 NIH/NIAAA, Rockville, MD, USA, 2NIH/NICHD, Bethesda, MD, USA. FRET experiments using Fluorescent proteins (FPs) typically assume that individual FPs act independently (i.e. weak coupling limit). However, several recent studies have observed that assemblies of FPs can act as a single photon source, indicating that FPs in these complexes interact in the strong coupling limit and may explain our observation of ultra-fast (<6 ps) energy transfer between FPs in Venus oligomers. To investigate the mechanism of this ultra-fast