Improved folding of apo-retinol-binding protein in the periplasm of Escherichia coli: positive influences of dsbC coexpression and of an amino acid exchange in the vitamin A binding site.
The in vivo folding of the serum retinol-binding protein (RBP), a representative of the lipocalin structural family, is known to be complex. In order to gain insight into the essential steps along its folding pathway the heterologous production of the functional protein in Escherichia coli was investigated. Simultaneous overexpression of the bacterial dsbC gene, which codes for a periplasmic thiol-disulphide oxidoreductase, prevented the formation of soluble RBP variants with non-native disulphide bonds that were otherwise observed. Although the coexpression of dsbC had furthermore a stabilizing effect on the cell viability, the relative yield of the solubly produced RBP was not much better. In an attempt to enhance its folding efficiency, a favourable point mutation in the inner part of the retinol-binding pocket was predicted. Replacement of the polar Gln117 with an lie side chain seemed not only to relieve the unfavourable energetics of the carboxamide group in the environment of predominantly non-polar residues but also to fill an adjacent cavity in the hydrophobic core. Indeed, this single substitution reproducibly resulted in a more than threefold increase in the amount of functional recombinant RBP. Ligand binding experiments showed that the affinity of this mutant for retinol was slightly enhanced. Kinetic measurements revealed that this was due to a higher association rate whereas the dissociation of the complex with retinol was essentially unaffected. Although the question remained why nature did not select this obviously beneficial mutation, our results demonstrate that the folding pathway of a lipocalin can be optimized by protein engineering.
Schmidt
A M
AM
Institut für Biochemie, Technische Universität, Darmstadt, Germany.
Bloss
I
I
Skerra
A
A
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases / Q000235:genetics; D017510:Protein Folding; D012177:Retinol-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D014801:Vitamin A / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D012177:Retinol-Binding Proteins; D014801:Vitamin A; D019704:Protein Disulfide-Isomerases