title: Single-site mutations in a hyperthermophilic variant of the B1 domain of protein G result in self-assembled oligomers.
abstract: We have characterized two homologous, single-point core mutants of a 57-residue, hyperthermophilic variant of the B1 domain of protein G (HTB1). These single-point mutations in HTB1 replace a Phe residue in the hydrophobic core with either a Glu or Asp residue. Both of these homologous core-variant mutants undergo significant structural rearrangement from the native, monomeric fold and exist as stable soluble oligomeric species of 5 and 30 nm in diameter. Gel-filtration, dynamic light scattering, circular dichroism spectroscopy, fluorescence spectroscopy, along with Congo Red and Thioflavin T binding clearly demonstrated that these core-variants undergo significant structural rearrangement from the native, monomeric ubiquitin fold. The two oligomeric species did not equilibrate over extended periods of time and displayed distinct secondary structures. The larger of the two species was found to possess structural features that are reminiscent of an emerging class of protein assemblies prone to beta-sheet-mediated aggregation. These results are significant as there are very few examples of extensive conformational or oligomerization switching brought about by single-point mutations in a stable protein-fold.
authors.affiliation: Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Meyer
authors.forename: Carmen
authors.initials: C
authors.lastname: Huerta
authors.forename: Indraneel
authors.initials: I
authors.lastname: Ghosh
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D052160:Benzothiazoles; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003224:Congo Red / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism; D014644:Genetic Variation; D006358:Hot Temperature; D007074:Immunoglobulin G / Q000378:metabolism; D008027:Light; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013844:Thiazoles / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D052160:Benzothiazoles; D005456:Fluorescent Dyes; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D013844:Thiazoles; C009462:thioflavin T; D003224:Congo Red