title: Stabilization of tryptophan hydroxylase 2 by l-phenylalanine-induced dimerization.
abstract: Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full-length TPH2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH2 (rchTPH2; regulatory and catalytic domain, NΔ47-rchTPH2; truncation of 47 residues in the N terminus of rchTPH2, and chTPH2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. chTPH2 displayed 14- and 11-fold higher half-lives compared to rchTPH2 and NΔ47-rchTPH2, respectively. Differential scanning calorimetry experiments demonstrated that this is caused by premature unfolding of the less stable regulatory domain. By differential scanning fluorimetry, the unfolding transitions of rchTPH2 and NΔ47-rchTPH2 are found to shift from polyphasic to apparent two-state by the addition of l-Trp or l-Phe. Analytical gel filtration revealed that rchTPH2 and NΔ47-rchTPH2 reside in a monomer-dimer equilibrium which is significantly shifted toward dimer in the presence of l-Phe. The dimerizing effect induced by l-Phe is accompanied by a stabilizing effect, which resulted in a threefold increase in half-lives of rchTPH2 and NΔ47-rchTPH2. Addition of l-Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization ofhTPH2.
authors.affiliation: Department of Chemistry Technical University of Denmark Kongens Lyngby Denmark.
authors.forename: Kasper D
authors.initials: KD
authors.lastname: Tidemand
authors.affiliation: Department of Chemistry Technical University of Denmark Kongens Lyngby Denmark.
authors.forename: Hans E M
authors.initials: HE
authors.lastname: Christensen
authors.affiliation: Department of Chemistry Technical University of Denmark Kongens Lyngby Denmark.
authors.forename: Niclas
authors.initials: N
authors.lastname: Hoeck
authors.affiliation: Department of Chemistry Technical University of Denmark Kongens Lyngby Denmark.
authors.forename: Pernille
authors.initials: P
authors.lastname: Harris
authors.affiliation: Department of Chemistry Technical University of Denmark Kongens Lyngby Denmark.
authors.forename: Jane
authors.initials: J
authors.lastname: Boesen
authors.affiliation: Department of Chemistry Technical University of Denmark Kongens Lyngby Denmark.
authors.forename: Günther H
authors.initials: GH
authors.lastname: Peters
keywords: analytical size exclusion chromatography; differential scanning fluorimetry; enzyme characterization; oligomerization; protein purification