@article{choi_mccann_weninger_bowen_2011, title={Beyond the Random Coil: Stochastic Conformational Switching in Intrinsically Disordered Proteins}, volume={19}, ISSN={["1878-4186"]}, DOI={10.1016/j.str.2011.01.011}, abstractNote={Intrinsically disordered proteins (IDPs) participate in critical cellular functions that exploit the flexibility and rapid conformational fluctuations of their native state. Limited information about the native state of IDPs can be gained by the averaging over many heterogeneous molecules that is unavoidable in ensemble approaches. We used single molecule fluorescence to characterize native state conformational dynamics in five synaptic proteins confirmed to be disordered by other techniques. For three of the proteins, SNAP-25, synaptobrevin and complexin, their conformational dynamics could be described with a simple semiflexible polymer model. Surprisingly, two proteins, neuroligin and the NMDAR-2B glutamate receptor, were observed to stochastically switch among distinct conformational states despite the fact that they appeared intrinsically disordered by other measures. The hop-like intramolecular diffusion found in these proteins is suggested to define a class of functionality previously unrecognized for IDPs.}, number={4}, journal={STRUCTURE}, author={Choi, Ucheor B. and McCann, James J. and Weninger, Keith R. and Bowen, Mark E.}, year={2011}, month={Apr}, pages={566–576} }
 @article{mccann_choi_zheng_weninger_bowen_2010, title={Optimizing Methods to Recover Absolute FRET Efficiency from Immobilized Single Molecules}, volume={99}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2010.04.063}, abstractNote={Microscopy-based fluorescence resonance energy transfer (FRET) experiments measure donor and acceptor intensities by isolating these signals with a series of optical elements. Because this filtering discards portions of the spectrum, the observed FRET efficiency is dependent on the set of filters in use. Similarly, observed FRET efficiency is also affected by differences in fluorophore quantum yield. Recovering the absolute FRET efficiency requires normalization for these effects to account for differences between the donor and acceptor fluorophores in their quantum yield and detection efficiency. Without this correction, FRET is consistent across multiple experiments only if the photophysical and instrument properties remain unchanged. Here we present what is, to our knowledge, the first systematic study of methods to recover the true FRET efficiency using DNA rulers with known fluorophore separations. We varied optical elements to purposefully alter observed FRET and examined protein samples to achieve quantum yields distinct from those in the DNA samples. Correction for calculated instrument transmission reduced FRET deviations, which can facilitate comparison of results from different instruments. Empirical normalization was more effective but required significant effort. Normalization based on single-molecule photobleaching was the most effective depending on how it is applied. Surprisingly, per-molecule gamma-normalization reduced the peak width in the DNA FRET distribution because anomalous gamma-values correspond to FRET outliers. Thus, molecule-to-molecule variation in gamma has an unrecognized effect on the FRET distribution that must be considered to extract information on sample dynamics from the distribution width.}, number={3}, journal={BIOPHYSICAL JOURNAL}, author={McCann, James J. and Choi, Ucheor B. and Zheng, Liqiang and Weninger, Keith and Bowen, Mark E.}, year={2010}, month={Aug}, pages={961–970} }
 @article{choi_strop_vrljic_chu_brunger_weninger_2010, title={Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex}, volume={17}, ISSN={["1545-9985"]}, DOI={10.1038/nsmb.1763}, abstractNote={Single-molecule FRET studies have resulted in an experimentally derived model of a synaptotagmin–SNARE complex. In this complex of SNARE with synaptotagmin 1, the arrangement of the Ca2+-binding loops is similar to that of the structure of SNARE-induced Ca2+-bound synaptotagmin 3. This suggests a common molecular mechanism by which the synaptotagmin–SNARE interaction plays a role in Ca2+-triggered vesicle fusion. Synchronous neurotransmission is triggered when Ca2+ binds to synaptotagmin 1 (Syt1), a synaptic-vesicle protein that interacts with SNAREs and membranes. We used single-molecule fluorescence resonance energy transfer (FRET) between synaptotagmin's two C2 domains to determine that their conformation consists of multiple states with occasional transitions, consistent with domains in random relative motion. SNARE binding results in narrower intrasynaptotagmin FRET distributions and less frequent transitions between states. We obtained an experimentally determined model of the elusive Syt1–SNARE complex using a multibody docking approach with 34 FRET-derived distances as restraints. The Ca2+-binding loops point away from the SNARE complex, so they may interact with the same membrane. The loop arrangement is similar to that of the crystal structure of SNARE-induced Ca2+-bound Syt3, suggesting a common mechanism by which the interaction between synaptotagmins and SNAREs aids in Ca2+-triggered fusion.}, number={3}, journal={NATURE STRUCTURAL & MOLECULAR BIOLOGY}, author={Choi, Ucheor B. and Strop, Pavel and Vrljic, Marija and Chu, Steven and Brunger, Axel T. and Weninger, Keith R.}, year={2010}, month={Mar}, pages={318–U84} }
 @article{weninger_bowen_choi_chu_brunger_2008, title={Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1 : 1 syntaxin/SNAP-25 complex}, volume={16}, ISSN={["1878-4186"]}, DOI={10.1016/j.str.2007.12.010}, abstractNote={Syntaxin/SNAP-25 interactions precede assembly of the ternary SNARE complex that is essential for neurotransmitter release. This binary complex has been difficult to characterize by bulk methods because of the prevalence of a 2:1 dead-end species. Here, using single-molecule fluorescence, we find the structure of the 1:1 syntaxin/SNAP-25 binary complex is variable, with states changing on the second timescale. One state corresponds to a parallel three-helix bundle, whereas other states show one of the SNAP-25 SNARE domains dissociated. Adding synaptobrevin suppresses the dissociated helix states. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex is a dynamic acceptor for synaptobrevin binding, and accessory proteins stabilize this acceptor. In the cellular environment the binary complex is actively maintained in a configuration where it can rapidly interact with synaptobrevin, so formation is not likely a limiting step for neurotransmitter release.}, number={2}, journal={STRUCTURE}, author={Weninger, Keith and Bowen, Mark E. and Choi, Ucheor B. and Chu, Steven and Brunger, Axel T.}, year={2008}, month={Feb}, pages={308–320} }