Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.

Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure,...

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Autores principales:	Simon Lindhoud, Adrie H Westphal, Jan Willem Borst, Carlo P M van Mierlo
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f23206c8ed51454bb2f166448be6d726
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spelling	oai:doaj.org-article:f23206c8ed51454bb2f166448be6d7262021-11-18T07:04:31ZIlluminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.1932-620310.1371/journal.pone.0045746https://doaj.org/article/f23206c8ed51454bb2f166448be6d7262012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23029219/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin's molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an α-β parallel protein.Simon LindhoudAdrie H WestphalJan Willem BorstCarlo P M van MierloPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 9, p e45746 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Simon Lindhoud Adrie H Westphal Jan Willem Borst Carlo P M van Mierlo Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.
description	Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin's molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an α-β parallel protein.
format	article
author	Simon Lindhoud Adrie H Westphal Jan Willem Borst Carlo P M van Mierlo
author_facet	Simon Lindhoud Adrie H Westphal Jan Willem Borst Carlo P M van Mierlo
author_sort	Simon Lindhoud
title	Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.
title_short	Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.
title_full	Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.
title_fullStr	Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.
title_full_unstemmed	Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.
title_sort	illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/f23206c8ed51454bb2f166448be6d726
work_keys_str_mv	AT simonlindhoud illuminatingtheoffpathwaynatureofthemoltenglobulefoldingintermediateofanabparallelprotein AT adriehwestphal illuminatingtheoffpathwaynatureofthemoltenglobulefoldingintermediateofanabparallelprotein AT janwillemborst illuminatingtheoffpathwaynatureofthemoltenglobulefoldingintermediateofanabparallelprotein AT carlopmvanmierlo illuminatingtheoffpathwaynatureofthemoltenglobulefoldingintermediateofanabparallelprotein
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Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.

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