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FEBS Open Bio. 2016 Aug 22;6(10):987-999. eCollection 2016 Oct.

Stabilization of tryptophan hydroxylase 2 by l-phenylalanine-induced dimerization.

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1
Department of Chemistry Technical University of Denmark Kongens Lyngby Denmark.

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 (rch TPH2; regulatory and catalytic domain, NΔ47-rch TPH2; truncation of 47 residues in the N terminus of rch TPH2, and ch TPH2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. ch TPH2 displayed 14- and 11-fold higher half-lives compared to rch TPH2 and NΔ47-rch TPH2, 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 rch TPH2 and NΔ47-rch TPH2 are found to shift from polyphasic to apparent two-state by the addition of l-Trp or l-Phe. Analytical gel filtration revealed that rch TPH2 and NΔ47-rch TPH2 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 rch TPH2 and NΔ47-rch TPH2. Addition of l-Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of hTPH2.

KEYWORDS:

analytical size exclusion chromatography; differential scanning fluorimetry; enzyme characterization; oligomerization; protein purification

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