title: Sml1p is a dimer in solution: characterization of denaturation and renaturation of recombinant Sml1p.
abstract: Sml1p is a small 104-amino acid protein from Saccharomyces cerevisiae that binds to the large subunit (Rnr1p) of the ribonucleotide reductase complex (RNR) and inhibits its activity. During DNA damage, S phase, or both, RNR activity must be tightly regulated, since failure to control the cellular level of dNTP pools may lead to genetic abnormalities, such as genome rearrangements, or even cell death. Structural characterization of Sml1p is an important step in understanding the regulation of RNR. Until now the oligomeric state of Sml1p was unknown. Mass spectrometric analysis of wild-type Sml1p revealed an intermolecular disulfide bond involving the cysteine residue at position 14 of the primary sequence. To determine whether disulfide bonding is essential for Sml1p oligomerization, we mutated the Cys14 to serine. Sedimentation equilibrium measurements in the analytical ultracentrifuge show that both wild-type and C14S Sml1p exist as dimers in solution, indicating that the dimerization is not a result of a disulfide bond. Further studies of several truncated Sml1p mutants revealed that the N-terminal 8-20 residues are responsible for dimerization. Unfolding/refolding studies of wild-type and C14S Sml1p reveal that both proteins refold reversibly and have almost identical unfolding/refolding profiles. It appears that Sml1p is a two-domain protein where the N-terminus is responsible for dimerization and the C-terminus for binding and inhibiting Rnr1p activity.
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, USA.
authors.forename: Vibha
authors.initials: V
authors.lastname: Gupta
authors.forename: Cynthia B
authors.initials: CB
authors.lastname: Peterson
authors.forename: Lezlee T
authors.initials: LT
authors.lastname: Dice
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Uchiki
authors.forename: Joseph
authors.initials: J
authors.lastname: Racca
authors.forename: Jun-tao
authors.initials: JT
authors.lastname: Guo
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.forename: Robert
authors.initials: R
authors.lastname: Hettich
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Zhao
authors.forename: Rodney
authors.initials: R
authors.lastname: Rothstein
authors.forename: Chris G
authors.initials: CG
authors.lastname: Dealwis
mesh_terms: D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D014461:Ultracentrifugation
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; C115052:SML1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D003545:Cysteine