title: Cysteine-free Rop: a four-helix bundle core mutant has wild-type stability and structure but dramatically different unfolding kinetics.
abstract: Cysteine residues can complicate the folding and storage of proteins due to improper formation of disulfide bonds or oxidation of residues that are natively reduced. Wild-type Rop is a homodimeric four-helix bundle protein and an important model for protein design in the understanding of protein stability, structure and folding kinetics. In the native state, Rop has two buried, reduced cysteine residues in its core, but these are prone to oxidation in destabilized variants, particularly upon extended storage. To circumvent this problem, we designed and characterized a Cys-free variant of Rop, including solving the 2.3 A X-ray crystal structure. We show that the C38A C52V variant has similar structure, stability and in vivo activity to wild-type Rop, but that it has dramatically faster unfolding kinetics like virtually every other mutant of Rop that has been characterized. This cysteine-free Rop has already proven useful for studies on solution topology and on the relationship of core mutations to stability. It also suggests a general strategy for removal of pairs of Cys residues in proteins, both to make them more experimentally tractable and to improve their storage properties for therapeutic or industrial purposes.
authors.affiliation: Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Sanjay B
authors.initials: SB
authors.lastname: Hari
authors.forename: Chang
authors.initials: C
authors.lastname: Byeon
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Lavinder
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Magliery
mesh_terms: D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D016601:RNA-Binding Proteins; D003545:Cysteine