Homodimers of mutant tryptophan synthase alpha-subunits in Escherichia coli.
Tryptophan synthase alpha-subunit from Escherichia coli functionally exists as a heterotetramer of alpha(2)beta(2) with beta-subunit. While wild-type and mutant (F139W, T24M/F139W, and T24L/F139W) alpha-subunits were expressed as a monomer from recombinant plasmids in Escherichia coli, T24A/F139W, T24S/F139W, and T24K/F139W mutant alpha-subunits were abnormally expressed as soluble homodimers in addition to monomers. Monomers of dimer-forming mutant alpha-subunits retain high affinity to beta-subunit, high activity in stimulating catalytic activities of beta-subunit, and nearly intact content of secondary structure, indicating that the global structures of these monomers are identical to that of F139W alpha-subunit. However, fluorescence spectra of Trp139 and ANS binding indicate that significant perturbations occur in the mutant proteins. Interestingly, these defective properties of monomers caused by residue replacement were partially repaired by the dimer formation. As a result, it is suggested that dimers may be formed by domain or loop swapping, and that residue 24 may play important role in maintaining on-pathway of alpha-subunit folding.
Kim
J W
JW
Department of Molecular Biology, College of Natural Sciences, Pusan National University, Pusan, 609-735, Korea.
Kim
E Y
EY
Park
H H
HH
Jung
J E
JE
Kim
H D
HD
Shin
H J
HJ
Lim
W K
WK
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005456:Fluorescent Dyes / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000235:genetics*; D014508:Urea / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D014367:Tryptophan Synthase