Synthesis of High Affinity Host-Guest FRET Pair and Synaptic Vesicle Fusion Study by Single-Vesicle Fusion Assay

Abstract

This thesis describes the synthesis and characterization of a high affinity host-guest fluorescence resonance energy transfer (FRET) pair and its application in single-vesicle fusion assay. The host-guest FRET pair, displays unique features such as small size, high affinity and high signal-to-noise ratio. These features then allow for the study of possible transient states of synaptic vesicle fusion which have not been previously observed in vitro and the dynamics involved in vesicle fusion process. Chapter 1 is a brief review of the recent developments in host-guest chemistry. Cucurbit[7]uril (CB[7]) and adamantylamine derivative (Ad-NHR) form a stable 1:1 host-guest complex with exceptionally high binding affinity (K ~ 1014 M-1) and selectivity, which makes the synthetic pair useful for high-fidelity bio-applications. Furthermore, this chapter highlights study of synaptic vesicle fusion mediated by a conserved family of proteins soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which is a key issue in neuronal communication. Chapter 2 presents the synthesis and characterization of a high-affinity fluorophore-tagged host-guest pair, CB[7]-Cy3 and Ad-Cy5, which generates a strong FRET signal upon host-guest complex formation. Monoamino-functionalized CB[7] was synthesized and conjugated with a donor dye Cy3 (CB[7]-Cy3). As the counterpart of CB[7]-Cy3, amine-tethered adamantane was prepared and subsequently conjugated with an acceptor dye Cy5 (Ad-Cy5). Both CB[7]-Cy3 and Ad-Cy5 were characterized by NMR spectroscopy and high resolution mass spectrometry. Furthermore, a high FRET signal in an aqueous solution upon host-guest binding of CB[7]-Cy3 and Ad-Cy5 was demonstrated. Chapter 3 describes a new, reliable, and efficient single-vesicle content-mixing assay for SNARE-mediated membrane fusion using a high affinity fluorophore tagged host-guest pair, CB[7]-Cy3 and Ad-Cy5 as a FRET pair. A change in FRET signal occurred when a CB[7]-Cy3 binds to a Ad-Cy5, which is a mark of two different SNARE embedded vesicle (CB[7]-Cy3@v-vesicle, Ad-Cy3@t-vesicle) fusion. Single-vesicle fusion assay based on fluorescence using total internal reflection fluorescence microscope (TIRF), in contrast to average characteristic measurements on an ensemble, enables us to monitor in real-time and distinguish multiple intermediates in the SNARE-mediated vesicle fusion between CB[7]-Cy3@v-vesicle and Ad-Cy5@t-vesicle. We discovered that our assay has the ability to distinguish between the docking and membrane fusion pore opening state with a high signal to noise ratio. Remarkably, CB[7]-Ad FRET pair with a high signal to noise ratio allows us to observe, for the first time, reversible fusion pore flickering events, i.e., a repetitive opening and closure of the fusion pore in an in vitro content-mixing assay. The results presented in this chapter demonstrate the potential of our novel content-mixing assay for the development of synaptic vesicle fusion study. Chapter 4 presents Ca2+ stimulation effects on the fusion pore flickering and its dynamics in single-vesicle fusion content-mixing assay based on a high affinity host-guest FRET pair. Ca2+ is known to trigger and accelerate the synaptic vesicle fusion. However, its stimulation effect on the reversible fusion pore flickering and its dynamics remain unknown. Our assay, for the first time, discovered that Ca2+ accelerates the fusion pore opening rate on the docking state and helps dilation of the fusion pore for fast and efficient neurotransmitter release at the initial pore opening step. However, it has no effect on the re-opening of fusion pore dynamics in the middle of the reversible fusion pore flickering. These results demonstrate that the content-mixing assay based on the high affinity host-guest FRET pair is a powerful tool to study the detailed vesicle fusion processes.