Productive interaction of chaperones with substrate protein domains allows correct folding of the downstream GFP domain.
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.
Zhang
Aihua
A
New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA.
Cantor
Eric J
EJ
Barshevsky
Tanya
T
Chong
Shaorong
S
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
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