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Direct Measurement of Sequence-Dependent Transition Path Times and Conformational Diffusion in DNA Duplex Formation
Wednesday, February 15, 2017 optical tweezers, we demonstrate that SNAREs zipper stage-wise with distinct kinetics. We directly observed four stages of assembly in SNARE N-terminal, middle, C-terminal, and linker domains (or NTD, MD, CTD and LD, respectively). Results of layer mutations suggest that NTD and CTD are responsible for vesicle docking and fusion, respectively, whereas MD regulates SNARE assembly and fusion. Munc18-1 intimately regulates SNARE assembly by initiating SNARE assembly and stabilizing the half-zippered SNARE complex. Our observations clarify the distinct functions of SNARE domains and the essential role of Munc 18-1 in synaptic exocytosis. We also developed novel assays to study membrane protein folding and stability based on optical tweezers. We applied the assays to folding dynamics of trans-SNAREs bridging two membranes and interactions of Syt and alpha-SNAP with membranes. 2316-Plat Direct Measurement of Sequence-Dependent Transition Path Times and Conformational Diffusion in DNA Duplex Formation Krishna Neupane1, Feng Wang2, Michael Woodside1. 1 Physics, University of Alberta, Edmonton, AB, Canada, 2National Institute for Nanotechnology, Edmonton, AB, Canada. The conformational diffusion coefficient, D, sets the timescale for microscopic structural changes during folding transitions in biomolecules like nucleic acids and proteins. D encodes significant information about the folding dynamics such as the roughness of the energy landscape governing the folding and the level of internal friction in the molecule, but it is challenging to measure. The most sensitive measure of D is the time required to cross the energy barrier that dominates folding kinetics, known as the transition path time. To investigate the sequence-dependence of D in DNA duplex formation, we measured individual transition paths from equilibrium folding trajectories of single DNA hairpins held under tension in high-resolution optical tweezers. Studying hairpins with the same helix length but with G:C base-pair content varying from 0-100%, we determined both the average time to cross the transition paths, ttp, and the distribution of individual transit times, PTP(t). We then estimated D from both ttp and PTP(t) from theories assuming one-dimensional diffusive motion over a harmonic barrier. ttp decreased roughly linearly with the G:C content of the hairpin helix, being 50% longer for hairpins with only A:T base-pairs than for those with only G:C base-pairs. Conversely, D increased linearly with helix G:C content, roughly doubling as the G:C content increased from 0-100%. These results reveal that G:C base-pairs form faster than A:T base-pairs because of faster conformational diffusion, possibly reflecting lower torsional barriers, and demonstrate the power of transition path measurements for elucidating the microscopic determinants of folding. 2317-Plat Direct Single-Molecule Measurements of Phycocyanobilin Photophysics in Monomeric C-Phycocyanin Allison H. Squires1, Quan Wang2, W.E. Moerner1. 1 Department of Chemistry, Stanford University, Stanford, CA, USA, 2 Lewis-Siegler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA. Phycobilisomes are highly organized pigment-protein antenna complexes found in cyanobacteria, rhodophyta, and cryptophytes that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms, and may even inform rational design of artificial light-harvesting systems. However, only allophycocyanin, a twopigment protein which forms the core of the phycobilisome, has been previously characterized at the single-molecule level (Wang and Moerner, PNAS 2015). Here, we present the first single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the mid-antenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single proteins in real-time, we directly monitor, in aqueous solution, the changing photophysical states of individual C-PC monomers from Spirulina platensis by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. We are able to resolve single-chromophore emission states for each of the three covalently bound phycocyanobilins (a-84, b-84, and b-155), providing the first direct photophysical characterization of these chemically identical molecules in intact proteins. Although some observed photophysical states of C-PC closely match the predictions of our theoretical FRET network model, other states exhibit a surprising quenching behavior which can be traced to the b-155 chromophore. These data suggest possible nondegenerate functional roles among the protein’s constituent pigments. (This research is supported by a grant from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy.)
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2318-Plat Simulation of FRET Dyes Allows Direct Comparison against Experimental Data Ines Reinartz, Claude Sinner, Alexander Schug. SCC, KIT, Eggenstein-Leopoldshafen, Germany. Single molecule Fo¨rster Resonance Energy Transfer (smFRET) experiments provide valuable insight into protein dynamics. Akin to a molecular ruler, different protein conformations can be observed by measuring the energy transfer depending on the distance between a donor and an acceptor fluorophore. Besides this distance, the energy transfer is also dependent on the mutual orientation of the dyes. Both can be gained from atomistic simulations [1]. Structure based models (SBMs) are based on energy landscape theory and the principle of minimal frustration [2,3]. With the help of eSBMTools [4] we integrate FRET dyes into an all-atom SBM. The computational efficiency of these simulation protocols allows simulating such processes on regular desktop computers. We developed a method to obtain FRET efficiency histograms from SBM simulations which are directly comparable to experimental measurements [unpublished]. With the distance and orientation distributions from our simulations, we want to improve the planning and interpretation of smFRET measurements. As an example, we compare distributions from 2-color and 3-color FRET experiments [5] and simulations [unpublished data] for ClyA in monomer and protomer conformation. [1] Hoefling, M., Lima, N., Haenni, D., Seidel, C. A. M., Schuler, B. and Grubm€uller, H., PLoS ONE 6, 2011. [2] Onuchic, J.N. and P.G. Wolynes, Curr. Opin. Struct. Bio. 14, 2004. [3] Schug, A. and J.N. Onuchic, Current opinion in pharmacology 10, 2010. [4] Lutz, B., C. Sinner, G. Heuermann, A. Verma, and A. Schug, Bioinformatics 29, 2013. [5] Benke, S., Roderer D., Wunderlich B., Nettels D., Glockshuber R. and Schuler B., Nat. Comm. 6, 2015. 2319-Plat Single-Molecule Peptide Fingerprinting Jetty van Ginkel, Mike Filius, Malwina Szczepaniak, Pawel Tulinski, Anne S. Meyer, Chirlmin Joo. BioNanoScience, Delft University of Technology, Delft, Netherlands. Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study such as single cell analysis unfeasible. Current detection limits also preclude proteomic analysis of low abundance proteins. We demonstrate the proof-of-concept of a single-molecule fluorescence peptide sequencing. We harness AAAþ protease ClpXP to linearize and scan proteins. By sequentially reading out fluorescence signals from labeled amino acids via FRET, we fingerprint the identity of a peptide. The repurposed ClpXP exhibits high processivity, uni-directional processing with a constant speed, and two orders of magnitude of dynamic range in sensitivity. It makes a promising approach to sequence full-length protein substrates using a small amount of sample. 2320-Plat An in Vitro Sample Generation Pipeline for High-Throughput SingleMolecule FRET Based Screening of Proteins Kambiz M. Hamadani1, Madeleine Jensen2, Wu Peng3, Jamie H.D. Cate4, Susan Marqusee2. 1 Chemistry and Biochemistry, California State University San Marcos, San Marcos, CA, USA, 2Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA, USA, 3Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA, 4Chemistry, University of California, Berkeley, Berkeley, CA, USA. Single-molecule methods access biomolecular distributions, transient states, and asynchronous dynamics inaccessible to standard ensemble techniques. Although extremely powerful, the ability to screen large biomolecular libraries using fluorescence-based single-molecule detection platforms is still a challenge due to the lack of high-throughput methods for the generation and screening of large libraries of dye-labeled proteins. Here, we demonstrate proof-of-principle that by combining purified and reconstituted in-vitro translation, quantitative unnatural amino acid incorporation via sense codon reassignment, and either strainpromoted or copper-catalyzed azide-alkyne cycloaddition we can overcome these bottlenecks. We present a purification-free and parallelizable in-vitro approach to generating dual-labeled proteins and ribosome-nascent-chain (RNC) libraries suitable for single-molecule FRET-based structural phenotyping. Importantly, dual-labeled RNC libraries enable single molecule co-localization of genotypes with phenotypes, and thus multiplexed single molecule screening of protein libraries (e.g. using zero-mode-waveguides). Such an approach to highthroughput single molecule screening may be useful for the in-vitro directed evolution of proteins with designer single molecule phenotypes.
471a
2318-Plat Simulation of FRET Dyes Allows Direct Comparison against Experimental Data Ines Reinartz, Claude Sinner, Alexander Schug. SCC, KIT, Eggenstein-Leopoldshafen, Germany. Single molecule Fo¨rster Resonance Energy Transfer (smFRET) experiments provide valuable insight into protein dynamics. Akin to a molecular ruler, different protein conformations can be observed by measuring the energy transfer depending on the distance between a donor and an acceptor fluorophore. Besides this distance, the energy transfer is also dependent on the mutual orientation of the dyes. Both can be gained from atomistic simulations [1]. Structure based models (SBMs) are based on energy landscape theory and the principle of minimal frustration [2,3]. With the help of eSBMTools [4] we integrate FRET dyes into an all-atom SBM. The computational efficiency of these simulation protocols allows simulating such processes on regular desktop computers. We developed a method to obtain FRET efficiency histograms from SBM simulations which are directly comparable to experimental measurements [unpublished]. With the distance and orientation distributions from our simulations, we want to improve the planning and interpretation of smFRET measurements. As an example, we compare distributions from 2-color and 3-color FRET experiments [5] and simulations [unpublished data] for ClyA in monomer and protomer conformation. [1] Hoefling, M., Lima, N., Haenni, D., Seidel, C. A. M., Schuler, B. and Grubm€uller, H., PLoS ONE 6, 2011. [2] Onuchic, J.N. and P.G. Wolynes, Curr. Opin. Struct. Bio. 14, 2004. [3] Schug, A. and J.N. Onuchic, Current opinion in pharmacology 10, 2010. [4] Lutz, B., C. Sinner, G. Heuermann, A. Verma, and A. Schug, Bioinformatics 29, 2013. [5] Benke, S., Roderer D., Wunderlich B., Nettels D., Glockshuber R. and Schuler B., Nat. Comm. 6, 2015. 2319-Plat Single-Molecule Peptide Fingerprinting Jetty van Ginkel, Mike Filius, Malwina Szczepaniak, Pawel Tulinski, Anne S. Meyer, Chirlmin Joo. BioNanoScience, Delft University of Technology, Delft, Netherlands. Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study such as single cell analysis unfeasible. Current detection limits also preclude proteomic analysis of low abundance proteins. We demonstrate the proof-of-concept of a single-molecule fluorescence peptide sequencing. We harness AAAþ protease ClpXP to linearize and scan proteins. By sequentially reading out fluorescence signals from labeled amino acids via FRET, we fingerprint the identity of a peptide. The repurposed ClpXP exhibits high processivity, uni-directional processing with a constant speed, and two orders of magnitude of dynamic range in sensitivity. It makes a promising approach to sequence full-length protein substrates using a small amount of sample. 2320-Plat An in Vitro Sample Generation Pipeline for High-Throughput SingleMolecule FRET Based Screening of Proteins Kambiz M. Hamadani1, Madeleine Jensen2, Wu Peng3, Jamie H.D. Cate4, Susan Marqusee2. 1 Chemistry and Biochemistry, California State University San Marcos, San Marcos, CA, USA, 2Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA, USA, 3Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA, 4Chemistry, University of California, Berkeley, Berkeley, CA, USA. Single-molecule methods access biomolecular distributions, transient states, and asynchronous dynamics inaccessible to standard ensemble techniques. Although extremely powerful, the ability to screen large biomolecular libraries using fluorescence-based single-molecule detection platforms is still a challenge due to the lack of high-throughput methods for the generation and screening of large libraries of dye-labeled proteins. Here, we demonstrate proof-of-principle that by combining purified and reconstituted in-vitro translation, quantitative unnatural amino acid incorporation via sense codon reassignment, and either strainpromoted or copper-catalyzed azide-alkyne cycloaddition we can overcome these bottlenecks. We present a purification-free and parallelizable in-vitro approach to generating dual-labeled proteins and ribosome-nascent-chain (RNC) libraries suitable for single-molecule FRET-based structural phenotyping. Importantly, dual-labeled RNC libraries enable single molecule co-localization of genotypes with phenotypes, and thus multiplexed single molecule screening of protein libraries (e.g. using zero-mode-waveguides). Such an approach to highthroughput single molecule screening may be useful for the in-vitro directed evolution of proteins with designer single molecule phenotypes.