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dc.date.accessioned | 2022-07-04T14:59:48Z | |
dc.date.available | 2022-07-04T14:59:48Z | |
dc.date.issued | 2014 | |
dc.identifier.uri | http://sedici.unlp.edu.ar/handle/10915/138794 | |
dc.description.abstract | 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. | en |
dc.format.extent | 248a-248a | es |
dc.language | en | es |
dc.subject | Parkinson’s disease | es |
dc.subject | Alpha-synuclein | es |
dc.title | Association of α-Synuclein with Lipid Vesicles | en |
dc.type | Articulo | es |
sedici.identifier.other | doi:10.1016/j.bpj.2013.11.1453 | es |
sedici.identifier.issn | 0006-3495 | es |
sedici.identifier.issn | 1542-0086 | es |
sedici.title.subtitle | Stopped-Flow Kinetics of Concerted Binding and Conformational Change | en |
sedici.creator.person | Jovin, Thomas M. | es |
sedici.creator.person | Shvadchak, Volodymyr V. | es |
sedici.creator.person | Siero, Remco | es |
sedici.creator.person | Falomir Lockhart, Lisandro Jorge | es |
sedici.creator.person | Subramaniam, Vinod | es |
sedici.subject.materias | Química | es |
sedici.subject.materias | Ciencias Exactas | es |
sedici.description.fulltext | true | es |
mods.originInfo.place | Instituto de Investigaciones Bioquímicas de La Plata | es |
sedici.subtype | Articulo | es |
sedici.rights.license | Creative Commons Attribution 4.0 International (CC BY 4.0) | |
sedici.rights.uri | http://creativecommons.org/licenses/by/4.0/ | |
sedici.description.peerReview | peer-review | es |
sedici.relation.journalTitle | Biophysical Journal | es |
sedici.relation.journalVolumeAndIssue | vol. 106, no. 2 | es |