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. Study holds ProTherm entries: 25701, 25702, 25703, 25704, 25705, 25706, 25707 Extra Details: Rop; four-helix bundle; cysteine-free; unfolding kinetics
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:56 p.m.
|Number of data points||34|
|Proteins||Regulatory protein rop ; Regulatory protein rop|
|Assays/Quantities/Protocols||Experimental Assay: dCp prot_conc:50 microM ; Experimental Assay: dG_H2O ; Experimental Assay: m ; Experimental Assay: dTm ; Experimental Assay: Cm ; Experimental Assay: dHvH prot_conc:50 microM ; Experimental Assay: dTm ; Experimental Assay: dCp prot_conc:50 microM ; Experimental Assay: Tm prot_conc:50 microM ; Experimental Assay: dHvH prot_conc:50 microM ; Experimental Assay: dCp prot_conc:2 mg/mL ; Experimental Assay: Tm prot_conc:2 mg/mL ; Experimental Assay: dHvH prot_conc:2 mg/mL ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHADELYRSCLARFGDDGENL|