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Biochemistry ; 56(50): 6575-6584, 2017 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-29168380


The fibrillation of misfolded neurodegenerative disease-related proteins has been extensively studied over the past few decades, but the initial oligomerization has been rarely examined even though some recent evidence indicated that small protein oligomers are more neurotoxic than long protein fibers. It is rather difficult to study the initial oligomerization (nucleation) because most experimental methods, such as the ß-sheet-related fluorescence assay and X-ray diffraction, are unable to detect small structureless protein oligomers. In this study, we have successfully developed a method to link a short thermally sensitive poly(N-isopropylacrylamide) (PNIPAM) chain to a model protein, Sup35NM, at a specific 31st residue site (Sup35NM-31m-PNIPAM) via the efficient thiol-ene Michael addition reaction. The oligomerization was studied by a combination of laser light scattering, the thioflavin T assay, and transmission electron microscopy. We found that the lag phase of Sup35NM was delayed from 12 to >24 h under the physiological condition after the PNIPAM linkage. The oligomerization and fibrillation constants decreased from 5.0 × 10-3 to 1.5 × 10-3 h-1 and from 3.0 × 10-2 to 1.8 × 10-2 µM-1 h-1, respectively, presumably because of the steric hindrance introduced by the PNIPAM chain. Moreover, after initiating the oligomerization, we found that the oligomer distribution in the first 6 h repeatedly and quantitatively follows the Smoluchowski coagulation model. Our study paves the way for controllably and quantitatively studying the oligomerization kinetics of amyloidogenic proteins. In addition, by investigating the effects of different small molecules on the oligomerization kinetics, we should be able to screen potential drugs to slow the development of neurodegenerative diseases.

Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Resinas Acrílicas/química , Resinas Acrílicas/metabolismo , Secuencia de Aminoácidos/genética , Cinética , Microscopía Electrónica de Transmisión , Factores de Terminación de Péptidos/fisiología , Proteínas Priónicas/química , Proteínas Priónicas/metabolismo , Priones/química , Unión Proteica/genética , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Difracción de Rayos X