title: The CBS domain protein MJ0729 of Methanocaldococcus jannaschii is a thermostable protein with a pH-dependent self-oligomerization.
abstract: CBS domains are small protein motifs, usually associated in tandems, that are involved in binding to adenosyl groups. In humans, several genetic diseases have been associated with mutations in CBS domains, and then, they can be considered as promising targets for the rational design of new drugs. However, there are no structural studies describing their oligomerization states, conformational preferences, and stability. In this work, the oligomerization state, the stability, and conformational properties of the CBS domain protein MJ0729 from Methanocaldococcus jannaschii were explored by using a combination of hydrodynamic (namely, ultracentrifugation, DLS, DOSY-NMR, and gel filtration) and spectroscopic techniques (fluorescence, circular dichroism, NMR, and FTIR). The results indicate that the protein had a pH-dependent oligomerization equilibrium: at pH 7, the dominant species is a dimer, where each monomer is a two-CBS domain protein, and at pH 4.5-4.8, the dominant species is a tetramer, with an oblong shape, as shown by X-ray. Deconvolution of the FTIR spectra indicates that the monomer at physiological pH has 26% alpha-helical structure and 17% beta-sheet, with most of the structure disordered. These results are similar to the percentages of secondary structure of the monomer in the resolved tetrameric X-ray structure (21% of alpha-helical structure and 7% of beta-sheet). At pH 2.5, there was a decrease in the level of secondary structure of the monomer, and formation of intermolecular hydrogen bonds, as shown by FTIR, suggesting the presence of high-molecular weight species. The physiological dimeric species is thermal and chemically very stable with a thermal midpoint of approximately 99 degrees C, as shown by both DSC and FTIR; the GdmCl chemical midpoint of the dimeric species occurs in a single step and was greater than 4 M.
authors.affiliation: Unidad de Biologia Estructural, CIC bioGUNE, Parque Tecnologico de Vizcaya, Derio, Bizkaia, Spain. amartinez@cicbiogune.es
authors.forename: Luis Alfonso
authors.initials: LA
authors.lastname: Martínez-Cruz
authors.forename: José A
authors.initials: JA
authors.lastname: Encinar
authors.forename: Danel
authors.initials: D
authors.lastname: Kortazar
authors.forename: Jesús
authors.initials: J
authors.lastname: Prieto
authors.forename: Javier
authors.initials: J
authors.lastname: Gómez
authors.forename: Pablo
authors.initials: P
authors.lastname: Fernández-Millán
authors.forename: María
authors.initials: M
authors.lastname: Lucas
authors.forename: Egoitz Astigarraga
authors.initials: EA
authors.lastname: Arribas
authors.forename: José Andrés
authors.initials: JA
authors.lastname: Fernández
authors.forename: María Luz
authors.initials: ML
authors.lastname: Martínez-Chantar
authors.forename: José M
authors.initials: JM
authors.lastname: Mato
authors.forename: José Luis
authors.initials: JL
authors.lastname: Neira
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D006863:Hydrogen-Ion Concentration; D017015:Methanococcales / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D019843:Archaeal Proteins