title: Methods for preparing crystals of reversibly oxidized proteins: crystallization of protein tyrosine phosphatase 1B as an example.
abstract: Regulation of protein activity through the oxidation and reduction of cysteines is emerging as an important mechanism in the control of cell-signaling pathways. Protein tyrosine phosphatase 1B (PTP1B), for example, is reversibly inhibited by oxidation at the catalytic cysteine in response to stimulation of cells by insulin or epidermal growth factor. We have conducted structural studies on the redox regulation of PTP1B and have demonstrated that the oxidation of the catalytic cysteine results in the formation of a bond between the sulfur atom of the catalytic cysteine and the amide nitrogen of the neighboring serine. This bond, referred to here as a sulfenamide bond, is reversible upon the addition of glutathione, indicating that this sulfenamide intermediate could function within signaling pathways to protect the cysteine from overoxidation to less readily reducible states. Formation of the sulfenamide bond is accompanied by changes in the tertiary structure at the catalytic site, and these changes may be important for additional regulation of the enzyme. Here, we present methods for preparing crystals ofPTP1B with a sulfenamide bond at the catalytic cysteine. The methods may be adaptable for other proteins that are subject to redox regulation.
authors.affiliation: Department of Chemical and Systems Biology, Stanford University Medical Center, Stanford, CA, USA.
authors.forename: Annette
authors.initials: A
authors.lastname: Salmeen
authors.forename: David
authors.initials: D
authors.lastname: Barford
mesh_terms: D020134:Catalytic Domain; D003460:Crystallization; D003545:Cysteine / Q000378:metabolism; D005615:Freezing; D008967:Molecular Biology / Q000379:methods*; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000737:chemistry / Q000378:metabolism*; D012996:Solutions
chemical_list: D012996:Solutions; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D003545:Cysteine