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43243-G4
Auto-Regulatory Conformational Dynamics in the Syntaxin Family of SNARE Proteins
Project overview
The goal of this project is to characterize the interactions that govern spontaneous conformational transitions in the syntaxin family of SNARE proteins. The SNARE proteins are small membrane proteins conserved in all species from yeast to humans that are essential for nearly all membrane fusion processes. Syntaxins, a SNARE sub-family, possess two distinct domains: an autonomously folded N-terminal domain and an adjacent domain involved in binding the partner SNAREs. Models of SNARE mediated membrane fusion posit that the N-terminal domain of syntaxin serves an auto-regulatory function, reversibly binding to syntaxin's SNARE binding domain to prevent the protein from forming the SNARE complex. When the N-terminal domain is bound to the SNARE binding domain, the protein is “closed” and cannot link with other SNAREs. When the domains are unbound, the protein is “open” to binding to other SNAREs to initiate membrane fusion.
The open-close model is the result of static structures observed in high-resolution studies, and this model is supported indirectly by biochemical experiments. We will directly confirm this model using the unique capability of fluorescence resonance energy transfer (FRET) to detect real-time intra-molecular conformational dynamics. To allow FRET measurements of the open-close transition, we have engineered specific labeling sites into syntaxin and sso1p (a syntaxin type SNARE from yeast) that allow attachment of two different fluorescent dyes. The first dye absorbs energy from a laser and can either emit red shifted light (fluorescence), or it can transfer energy to the second dye in a strongly distance dependent manner. Energy transferred to the second dye is emitted at a different color. By measuring the relative intensity of the emitted colors, FRET provides a ruler for measuring the dynamic motion of the proteins on the nanometer scale.
Knowledge of the basic processes regulating membrane fusion will lead to advances in our understanding of a wide variety of biological processes as SNAREs are essential for many processes including neurotransmitter release, insulin secretion and basic cellular metabolism. The methods we develop here will be directly relevant for studies of the spontaneous motions of other macromolecules.
Summary of results
One physics graduate student and two undergraduate physics students have worked on this project. In the first year of this project, we successfully purified the SNARE proteins syntaxin1A and sso1p using recombinant expression in e. coli. We also attached fluorescent dyes to specific labeling sites engineered into the SNARE binding domain and the regulatory domain of these proteins. The labeling sites were selected to yield high FRET (near 1) when the protein is closed and low FRET (near 0) when the protein is open. Single proteins were immobilized at a surface by encapsulating them inside liposomes that are tethered to a quartz slide through a biotin-streptavidin interaction. A wide field, total internal reflection fluorescence microscope measured single molecule FRET from these constructs. We observed emission from syntaxin and sso1p with FRET values of 0, ˝ and 1 and also transitions between these states. We interpret the FRET ˝ state averaging of the FRET 0 and FRET 1 states due to Brownian motion of the N-terminal domain in the open conformation that intermittently brings the two dyes into close proximity. At the time resolution of our wide field microscope (100 msec), a rapid open/close switching averages between FRET 1 and 0 and yield FRET ˝.
We have verified our interpretation of the FRET ˝ state as rapid averaging of a FRET 0 and FRET 1 state by constructing a scanning, confocal microscope based on commercial avalanche photodiodes for light detection to improve the time resolution. With this new instrument we have made real-time FRET measurements of single molecules with signal to noise of 3 and time resolution of 100 microseconds. This instrument has allowed us to directly observe rapid switching between high and low FRET states for one mutant of sso1p.
In the second year of this project we compared the open-close transition kinetics in syntaxin and sso1p, and mutants of these proteins designed to disrupt the binding interface between the two domains. We also designed new labeling sites into these SNARE proteins that provided distinct levels of FRET for these states. Our new constructs allowed us to more precisely determine the overall conformation of the proteins. Obtaining results consistent with the previous labeling sites when using the new labeling sites also confirms that dye labeling does not interfere with the natural kinetics of the proteins. We have compared the open-close switching of syntaxin in isolation to the switching of the protein when incorporated in binary complex with SNAP25 or in ternary SNARE complex when combined with both SNAP25 and synaptobrevin. We are presently investigating the influence of several other proteins known to regulate neurotransmission on the open-close equilibrium of neuronal syntaxin.
