Keith Weninger

Defining the unknowns of sonoluminescence

Abstract As the intensity of a standing sound wave is increased the pulsations of a bubble of gas trapped at a velocity node attain sufficient amplitude so as to emit picosecond flashes of light with a broadband spectrum that increases into the ultraviolet. The acoustic resonator can be tuned so that the flashes of light occur with a clocklike regularity: one flash for each cycle of sound with a jitter in the time between flashes that is also measured in picoseconds. This phenomenon (sonoluminescence or “SL”) is remarkable because it is the only means of generating picosecond flashes of light that does not use a laser and the input acoustic energy density must be concentrated by twelve orders of magnitude in order to produce light. Light scattering measurements indicate that the bubble wall is collapsing at more than 4 times the ambient speed of sound in the gas just prior to the light emitting moment when the gas has been compressed to a density determined by its van der Waals hard core. Experiments indicate that the collapse is remarkably spherical, water is the best fluid for SL, some noble gas is essential for stable SL, and that the light intensity increases as the ambient temperature is lowered. In the extremely stable experimental configuration consisting of an air bubble in water, measurements indicate that the bubble chooses an ambient radius that is not explained by mass diffusion. Experiments have not yet been able to map out the complete spectrum because above 6 eV it is obscured by the cutoff imposed by water, and furthermore experiments have only determined an upper bound on the flash widths. In addition to the above puzzles, the theory for the light emitting mechanism is still open. The scenario of a supersonic bubble collapse launching an imploding shock wave which ionizes the bubble contents so as to cause it to emit Bremsstrahlung radiation is the best candidate theory but it has not been shown how to extract from it the richness of this phenomenon. Most exciting is the issue of whether SL is a classical effect or whether Plancks constant should be invoked to explain how energy which enters a medium at the macroscopic scale holds together and focuses so as to be emitted at the microscopic scale.

Single-molecule FRET–derived model of the synaptotagmin 1–SNARE fusion complex

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 synaptotagmins 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.

Accessory Proteins Stabilize the Acceptor Complex for Synaptobrevin, the 1:1 Syntaxin/SNAP-25 Complex

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.

Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations

Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, synaptobrevin, and synaptosome-associated protein of 25 kDa on deposited lipid bilayers. Surprisingly, we found a mixture of parallel as well as antiparallel configurations involving the SNARE motifs of syntaxin and synaptobrevin as well as those of syntaxin and synaptosome-associated protein of 25 kDa. The subpopulation with the parallel four-helix bundle configuration could be greatly enriched by an additional purification step in the presence of denaturant, indicating that the parallel configuration is the energetically most favorable state. Interconversion between the configurations was not observed. From this observation, we infer the conversion rate to be <1.5 h–1. The existence of antiparallel configurations suggests a regulatory role of chaperones, such as N-ethylmaleimide-sensitive factor, or the membrane environment during SNARE complex assembly in vivo, and it could be a partial explanation for the relatively slow rates of vesicle fusion observed by reconstituted fusion experiments in vitro.

Detecting the conformation of individual proteins in live cells

We combined single-molecule fluorescence resonance energy transfer (smFRET) with single-particle tracking in live cells to detect the in vivo conformation of individual proteins. We site-specifically labeled recombinant SNARE proteins with a FRET donor and acceptor before microinjecting them into cultured cells. Individual proteins rapidly incorporated into folded complexes at the cell membrane, demonstrating the potential of this method to reveal dynamic interactions within cells.

Effect of Noble Gas Doping in Single-Bubble Sonoluminescence

The trillionfold concentration of sound energy by a trapped gas bubble, so as to emit picosecond flashes of ultraviolet light, is found to be extremely sensitive to doping with a noble gas. Increasing the noble gas content of a nitrogen bubble to about 1% dramatically stabilizes the bubble motion and increases the light emission by over an order of magnitude to a value that exceeds the sonoluminescence of either gas alone. The spectrum also strongly depends on the nature of the gas inside the bubble: Xenon yields a spectral peak at about 300 nanometers, whereas the helium spectrum is so strongly ultraviolet that its peak is obscured by the cutoff of water.

Single-Molecule Studies of the Neuronal SNARE Fusion Machinery

SNAREs are essential components of the machinery for Ca(2+)-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca(2+) sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.

Three-dimensional molecular modeling with single molecule FRET.

Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.

Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signalling

MutS protein recognizes mispaired bases in DNA and targets them for mismatch repair. Little is known about the transient conformations of MutS as it signals initiation of repair. We have used single‐molecule fluorescence resonance energy transfer (FRET) measurements to report the conformational dynamics of MutS during this process. We find that the DNA‐binding domains of MutS dynamically interconvert among multiple conformations when the protein is free and while it scans homoduplex DNA. Mismatch recognition restricts MutS conformation to a single state. Steady‐state measurements in the presence of nucleotides suggest that both ATP and ADP must be bound to MutS during its conversion to a sliding clamp form that signals repair. The transition from mismatch recognition to the sliding clamp occurs via two sequential conformational changes. These intermediate conformations of the MutS:DNA complex persist for seconds, providing ample opportunity for interaction with downstream proteins required for repair.

Beyond the Random Coil: Stochastic Conformational Switching in Intrinsically Disordered Proteins

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.