title: Productive interaction of chaperones with substrate protein domains allows correct folding of the downstream GFP domain.
abstract: Green fluorescent protein (GFP) has been used to report protein folding by correlating solubility with fluorescence. In a GFP fusion protein, an upstream aggregation-prone domain can disrupt de novo folding of the GFP domain in Escherichia coli, resulting in a loss of fluorescence. Previously, we showed that prevention of misfolding of the upstream aggregation-prone domain by a coupled folding and binding interaction during protein synthesis restored both GFP fluorescence and solubility. Since molecular chaperones often fold nascent polypeptides through a bind-and-release interaction, the question remains whether the chaperone interaction with the upstream aggregation-prone domain enhances GFP fluorescence. Here, we demonstrate that a significant increase in GFP fluorescence occurred only when appropriate chaperones that recognized the aggregation-prone protein and helped its folding were co-expressed. A possible correlation between GFP fluorescence and the productive folding by chaperones is proposed. This study may provide a general strategy for identifying chaperones specific for difficult-to-fold proteins.
authors.affiliation: New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA.
authors.forename: Aihua
authors.initials: A
authors.lastname: Zhang
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Cantor
authors.forename: Tanya
authors.initials: T
authors.lastname: Barshevsky
authors.forename: Shaorong
authors.initials: S
authors.lastname: Chong
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010749:Phosphoprotein Phosphatases; C063842:dnaK protein, E coli