Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.

The biophysical stability is an important parameter for protein activity both in vivo and in vitro. Here we propose a method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp). Combining unfolding by a temperature gradient in a thermal cycler with simultaneo...

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Autores principales:	David P Minde, Madelon M Maurice, Stefan G D Rüdiger
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8434dd8b31c14f2597496c289286d9e6
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spelling	oai:doaj.org-article:8434dd8b31c14f2597496c289286d9e62021-11-18T08:13:23ZDetermining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.1932-620310.1371/journal.pone.0046147https://doaj.org/article/8434dd8b31c14f2597496c289286d9e62012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23056252/?tool=EBIhttps://doaj.org/toc/1932-6203The biophysical stability is an important parameter for protein activity both in vivo and in vitro. Here we propose a method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp). Combining unfolding by a temperature gradient in a thermal cycler with simultaneous proteolytic cleavage of the unfolded state, we probed stability of single domains in lysates. We validated FASTpp on proteins from 10 kDa to 240 kDa and monitored stabilisation and coupled folding and binding upon interaction with small-molecule ligands. Within a total reaction time of approximately 1 min, we probed subtle stability differences of point mutations with high sensitivity and in agreement with data obtained by intrinsic protein fluorescence. We anticipate a wide range of applications of FASTpp in biomedicine and protein engineering as it requires only standard laboratory equipment.David P MindeDavid P MindeMadelon M MauriceStefan G D RüdigerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 10, p e46147 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q David P Minde David P Minde Madelon M Maurice Stefan G D Rüdiger Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.
description	The biophysical stability is an important parameter for protein activity both in vivo and in vitro. Here we propose a method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp). Combining unfolding by a temperature gradient in a thermal cycler with simultaneous proteolytic cleavage of the unfolded state, we probed stability of single domains in lysates. We validated FASTpp on proteins from 10 kDa to 240 kDa and monitored stabilisation and coupled folding and binding upon interaction with small-molecule ligands. Within a total reaction time of approximately 1 min, we probed subtle stability differences of point mutations with high sensitivity and in agreement with data obtained by intrinsic protein fluorescence. We anticipate a wide range of applications of FASTpp in biomedicine and protein engineering as it requires only standard laboratory equipment.
format	article
author	David P Minde David P Minde Madelon M Maurice Stefan G D Rüdiger
author_facet	David P Minde David P Minde Madelon M Maurice Stefan G D Rüdiger
author_sort	David P Minde
title	Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.
title_short	Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.
title_full	Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.
title_fullStr	Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.
title_full_unstemmed	Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.
title_sort	determining biophysical protein stability in lysates by a fast proteolysis assay, fastpp.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/8434dd8b31c14f2597496c289286d9e6
work_keys_str_mv	AT davidpminde determiningbiophysicalproteinstabilityinlysatesbyafastproteolysisassayfastpp AT davidpminde determiningbiophysicalproteinstabilityinlysatesbyafastproteolysisassayfastpp AT madelonmmaurice determiningbiophysicalproteinstabilityinlysatesbyafastproteolysisassayfastpp AT stefangdrudiger determiningbiophysicalproteinstabilityinlysatesbyafastproteolysisassayfastpp
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Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.

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