Destabilizing interactions between the partners of a bifunctional fusion protein.
Hybrid MalE-GVP is a bifunctional protein in vitro since it binds maltose as protein MalE of Escherichia coli and since it is dimeric and specifically binds single-stranded DNA as protein GVP of phage M13. The oxidation rate of a unique cysteine residue was used to compare the stabilities of GVP in its free and hybrid forms, under conditions where MalE was either folded or unfolded by a denaturing agent. The results showed that both the covalent link and tertiary non-covalent interactions between MalE and GVP destabilized GVP in MalE-GVP. To test whether GVP had identical structures in its free and hybrid forms, mutations were used as local conformational probes. The effects of these mutations on the capabilities of MalE-GVP to dimerize and to bind single-stranded DNA were assayed in vitro. They were compatible with the effects of the same mutations on the global activity of free GVP in vivo and with the effects that could be predicted from the known data on free GVP, in particular its crystal structure. Thus, one partner of a hybrid protein can be destabilized by the other partner while maintaining its structural and functional characteristics.
Blondel
A
A
CNRS URA 1129, Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
Nageotte
R
R
Bedouelle
H
H
D018528:ATP-Binding Cassette Transporters; D017104:Bacteriophage M13 / Q000737:chemistry; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D033902:Periplasmic Binding Proteins; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014461:Ultracentrifugation
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D006146:Guanidines; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D004228:Dithionitrobenzoic Acid; D019791:Guanidine; D003545:Cysteine