RESUMEN
In this chapter, we introduce a nanodisc-based experimental platform to study Ca2+-triggered membrane interaction of synaptotagmin-1. We describe and discuss in detail how to assemble this soluble mimetic of the docked vesicle-plasma membrane junction, with fluorescently labeled synaptotagmin-1 bound to trans SNAREpins assembled between nanodiscs and present the stopped-flow rapid mixing method used to monitor the conformational dynamics of Ca2+-activation process on a millisecond timescale.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Nanoestructuras/química , Sinaptotagmina I/metabolismo , Calcio/metabolismo , Cisteína/genética , Colorantes Fluorescentes/química , Membrana Dobles de Lípidos/química , Fusión de Membrana , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis Espectral/instrumentación , Análisis Espectral/métodos , Proteína 25 Asociada a Sinaptosomas/química , Proteína 25 Asociada a Sinaptosomas/aislamiento & purificación , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagmina I/química , Sinaptotagmina I/genética , Sinaptotagmina I/aislamiento & purificación , Sintaxina 1/química , Sintaxina 1/aislamiento & purificación , Sintaxina 1/metabolismoRESUMEN
Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+ Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery.
Asunto(s)
Calcio/metabolismo , Exocitosis , Fusión de Membrana/fisiología , Multimerización de Proteína/fisiología , Sinaptotagmina I/metabolismo , Animales , Cationes Bivalentes/metabolismo , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestructura , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/química , Microscopía Electrónica , Mutación , Células PC12 , Unión Proteica/fisiología , Ratas , Proteínas Recombinantes/metabolismo , Sinaptotagmina I/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Arginine methylation is important in biological systems. Recent studies link the deregulation of protein arginine methyltransferases with certain cancers. To assess the impact of methylation on interaction with other biomolecules, the pKa values of methylated arginine variants were determined using NMR data. The pKa values of monomethylated, symmetrically dimethylated, and asymmetrically dimethylated arginine are similar to the unmodified arginine (14.2 ± 0.4). Although the pKa value has not been significantly affected by methylation, consequences of methylation include changes in charge distribution and steric effects, suggesting alternative mechanisms for recognition.