Topological mutation of Escherichia coli dihydrofolate reductase.
Proteins appear to contain structural elements which determine the folded structure. If such elements are present, the order of structural elements in the primary structure, i.e. the chain topology, can be disregarded for building of the folded tertiary structure, when they are properly connected to each other by proper linkers. To experimentally examine this, "topological" mutants (designated as GHF33 and GHF34) of Escherichia coli dihydrofolate reductase (DHFR) were designed and constructed by switching two amino acid sequence parts containing the betaF strand and betaG-betaH strands in the primary sequence. In this way, the chain topology of wild-type DHFR, betaA-->alphaB-->betaB-->alphaC-->betaC--> betaD-->alphaE-->betaE-->al phaF-->betaF-->betaG-->betaH, was changed to betaA-->alphaB-->betaB-->alphaC-->betaC--> betaD-->alphaE-->betaE-->al phaF-->betaG-->betaH-->betaF. Such topological mutant proteins were stably expressed and accumulated in E. coli cells, and highly purified. Molecular mass measurements of the purified proteins and their proteolytic fragments confirmed that GHF33 and GHF34 had the designed sequence. In terms of kcat, the GHF33 and GHF34 proteins showed about 10 and 20% of the DHFR activity of the wild-type with Km values of 3.3 microM (GHF33) and 2.1 microM (GHF34), respectively. The topological mutants showed a cooperative two-state transition in urea-induced unfolding experiments with DeltaGH2O values of 4.0 and 4.8 kcal/mol. Whereas, the Km and DeltaGH2O values for wild-type DHFR were 0.9 microM and 6.1 kcal/mol, respectively. The significance of the topological mutations was discussed with respect to protein folding and protein evolution.
Iwakura
M
M
National Institute of Bioscience and Human-Technology, Tsukuba, Ibaraki, 305-8566, Japan. iwakura@nibh.go.jp
Takenawa
T
T
Nakamura
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase