Tertiary structure-dependence of misfolding substitutions in loops of the maltose-binding protein.
We previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the alphaI/betaB loop, of the N-domain that are critical for in vivo folding of the maltose-binding protein (MalE31). The tertiary context-dependence of this mutation in MalE folding was assessed by probing the tolerance of an equivalent alphabeta loop of the C-domain to the same amino acid substitutions (MalE219). Moving the loop mutation from the N- to the C-domain eliminated the in vivo misfolding step that led to the formation of inclusion bodies. In vitro, both loop variants exhibited an important decrease of stability, but their intrinsic tendency to aggregate was well correlated with their periplasmic fates in Escherichia coli. Furthermore, the noncoincidence of the unfolding and refolding transition curves and increase of light scattering during the refolding of MalE31 indicate that a competing off-pathway reaction could occurs on the folding pathway of this variant. These results strongly support the notion that the formation of super-secondary structures of the N-domain is a rate-limiting step in the folding pathway of MalE.
Raffy
S
S
Unité de Programmation Moléculaire et de Toxicologie Génétique/CNRS-URA1444, Département des Biotechnologies, Institut Pasteur, Paris, France.
Sassoon
N
N
Hofnung
M
M
Betton
J M
JM
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D033902:Periplasmic Binding Proteins; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C461599:maltose transport system, E coli; D019791:Guanidine