Association of α-Synuclein with Lipid Vesicles : Stopped-Flow Kinetics of Concerted Binding and Conformational Change

Alpha-synuclein (AS), a 140aa intrinsically disordered protein, self-associates into oligomeric forms and aggregates into amyloid fibrils in Parkinson's disease. Certain mutations affect these processes and accelerate disease pathogenesis. The physiological roles of AS are a matter of speculati...

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Autores principales: Jovin, Thomas M., Shvadchak, Volodymyr V., Siero, Remco, Falomir Lockhart, Lisandro Jorge, Subramaniam, Vinod
Formato: Articulo
Lenguaje:Inglés
Publicado: 2014
Materias:
Química
Ciencias Exactas
Parkinson’s disease
Alpha-synuclein
Acceso en línea:http://sedici.unlp.edu.ar/handle/10915/138794
Aporte de:
SEDICI (UNLP) de Universidad Nacional de La Plata
id I19-R120-10915-138794
record_format dspace
institution Universidad Nacional de La Plata
institution_str I-19
repository_str R-120
collection SEDICI (UNLP)
language Inglés
topic Química
Ciencias Exactas
Parkinson’s disease
Alpha-synuclein
spellingShingle Química
Ciencias Exactas
Parkinson’s disease
Alpha-synuclein
Jovin, Thomas M.
Shvadchak, Volodymyr V.
Siero, Remco
Falomir Lockhart, Lisandro Jorge
Subramaniam, Vinod
Association of α-Synuclein with Lipid Vesicles : Stopped-Flow Kinetics of Concerted Binding and Conformational Change
topic_facet Química
Ciencias Exactas
Parkinson’s disease
Alpha-synuclein
description Alpha-synuclein (AS), a 140aa intrinsically disordered protein, self-associates into oligomeric forms and aggregates into amyloid fibrils in Parkinson's disease. Certain mutations affect these processes and accelerate disease pathogenesis. The physiological roles of AS are a matter of speculation. Membrane binding is undoubtedly involved and the protein acquires α-helical structure in the process (1).We have studied the thermodynamics and kinetics of AS-membrane association utilizing vesicles (SUVs) of differing composition. Functionally neutral single cysteine mutants of AS were labeled with a polarity sensitive excited-state intramolecular proton transfer (ESIPT) probe (MFE). Double cysteine mutants were labeled with a FRET pair (Alexa Fluor488, Alexa Fluor568) at a series of selected positions in the primary sequence. Kinetic studies were conducted by stopped-flow, using 5-20 nM protein concentrations and increasing levels of SUVs (generally 20-200 µM) Signal changes indicative of membrane association were observed: increased intensity and shape change of dual band ESIPT emission, and altered FRET with the Alexa dyes. The analysis revealed a two-step reaction sequence in the time range <10 s. We attribute the first step to binding, and from the dependence on lipid concentration determined the second order rate constants and corresponding spectroscopic parameters. The second concentration independent step (1-10 s range) presumably arises from conformational changes in the protein (α-helix formation) and its accommodation to or perturbation of the lipid microenvironment (ESIPT dye).Accompanying thermodynamic measurements led to estimates of dissociation constants as a function of membrane composition, charge, and shape (SUVs, LUVs). A new experimental protocol (slopes), implemented in a microplate reader, circumvented technical problems usually manifested in titrations of protein with lipid.
format Articulo
Articulo
author Jovin, Thomas M.
Shvadchak, Volodymyr V.
Siero, Remco
Falomir Lockhart, Lisandro Jorge
Subramaniam, Vinod
author_facet Jovin, Thomas M.
Shvadchak, Volodymyr V.
Siero, Remco
Falomir Lockhart, Lisandro Jorge
Subramaniam, Vinod
author_sort Jovin, Thomas M.
title Association of α-Synuclein with Lipid Vesicles : Stopped-Flow Kinetics of Concerted Binding and Conformational Change
title_short Association of α-Synuclein with Lipid Vesicles : Stopped-Flow Kinetics of Concerted Binding and Conformational Change
title_full Association of α-Synuclein with Lipid Vesicles : Stopped-Flow Kinetics of Concerted Binding and Conformational Change
title_fullStr Association of α-Synuclein with Lipid Vesicles : Stopped-Flow Kinetics of Concerted Binding and Conformational Change
title_full_unstemmed Association of α-Synuclein with Lipid Vesicles : Stopped-Flow Kinetics of Concerted Binding and Conformational Change
title_sort association of α-synuclein with lipid vesicles : stopped-flow kinetics of concerted binding and conformational change
publishDate 2014
url http://sedici.unlp.edu.ar/handle/10915/138794
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