Solubilizing buried domains of proteins: a self-assembling interface domain from glutathione reductase.
In the dimeric glutathione reductase (GR) from Escherichia coli, the interface domain is largely surrounded by the other three domains in each subunit of the protein. Subgenes encoding three forms of the interface domain have been expressed in E. coli and the products purified from inclusion bodies: INT is the excised interface domain, as it is found in native GR; INTN and INTFN are variants carrying exchanges of surface residues in what would have been hydrophobic contact regions with other neighboring domains. The isolated INT domain was found to be a soluble and folded protein, but it was isolated as a mixture of the dimer and at least two species of higher molecular weight. The latter were believed to arise by further association of the dimer via the newly exposed and unsatisfied hydrophobic contact regions. In the variant INTN, three hydrophobic residues normally involved in the contact with the NADPH-binding domain in GR were replaced. This partly suppressed the further aggregation of the dimers. However, continued aggregation at high protein concentrations suggested that at least one further site of unwanted aggregation was still present. After four additional amino acid replacements in the region normally in contact with the FAD-binding domain, the resulting variant INTFN exhibited no unspecific aggregation, even at concentrations as high as 3.2 mg/mL.(ABSTRACT TRUNCATED AT 250 WORDS)
Leistler
B
B
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, U.K.
Perham
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RN
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017931:DNA Primers; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005980:Glutathione Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility
D017931:DNA Primers; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005980:Glutathione Reductase