title: Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome.
abstract: Correct folding of proteins is crucial for cellular homeostasis. More than thirty percent of proteins contain one or more cofactors, but the impact of these cofactors on co-translational folding remains largely unknown. Here, we address the binding of flavin mononucleotide (FMN) to nascent flavodoxin, by generating ribosome-arrested nascent chains that expose either the entire protein or C-terminally truncated segments thereof. The native α/β parallel fold of flavodoxin is among the most ancestral and widely distributed folds in nature and exploring its co-translational folding is thus highly relevant. In Escherichia coli (strain BL21(DE3) Δtig::kan) FMN turns out to be limiting for saturation of this flavoprotein on time-scales vastly exceeding those of flavodoxin synthesis. Because the ribosome affects protein folding, apoflavodoxin cannot bind FMN during its translation. As a result, binding of cofactor to released protein is the last step in production of this flavoprotein in the cell. We show that once apoflavodoxin is entirely synthesized and exposed outside the ribosome to which it is stalled by an artificial linker containing the SecM sequence, the protein is natively folded and capable of binding FMN.
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands.
authors.forename: Joseline A
authors.initials: JA
authors.lastname: Houwman
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands.
authors.forename: Adrie H
authors.initials: AH
authors.lastname: Westphal
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands.
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands. Electronic address: carlo.vanmierlo@wur.nl.
authors.forename: Carlo P M
authors.initials: CP
authors.lastname: van Mierlo
mesh_terms: D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D016948:Azotobacter vinelandii / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D008958:Models, Molecular; D011485:Protein Binding; D014176:Protein Biosynthesis; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D005418:Flavodoxin; D011994:Recombinant Proteins; C026932:apoflavodoxin; D005486:Flavin Mononucleotide
keywords: Co-translational protein folding; Cofactor binding; Flavin mononucleotide (FMN); Protein synthesis; Ribosome-nascent chain complex; Translational stalling