Stabilization of Tryptophan Hydroxylase 2 by L-Phenylalanine Induced Dimerization

Kasper Damgaard Tidemand, Hans Erik Mølager Christensen, Niclas Hoeck, Pernille Harris, Jane Boesen, Günther H.J. Peters

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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 towards dimer in the presence of L-Phe. The dimerizing effect induced by L-Phe is accompanied by a stabilizing effect, which resulted in a 3-fold 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 of hTPH2.
Original languageEnglish
JournalF E B S Open Bio
Volume6
Issue number10
Pages (from-to)987-999
Number of pages13
ISSN2211-5463
DOIs
Publication statusPublished - 2016

Bibliographical note

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Keywords

  • Analytical size exclusion chromatograph
  • Differential scanning fluorimetry
  • Enzyme characterization
  • Oligomerization
  • Protein purification

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