Journal article
Stabilization of Tryptophan Hydroxylase 2 by L-Phenylalanine Induced Dimerization
Department of Chemistry, Technical University of Denmark1
Metalloprotein Chemistry and Engineering, Department of Chemistry, Technical University of Denmark2
Organic Chemistry, Department of Chemistry, Technical University of Denmark3
X-ray Crystallography, Department of Chemistry, Technical University of Denmark4
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.
Language: | English |
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Publisher: | John Wiley and Sons Inc. |
Year: | 2016 |
Pages: | 987-999 |
ISSN: | 22115463 |
Types: | Journal article |
DOI: | 10.1002/2211-5463.12100 |
ORCIDs: | Christensen, Hans Erik Mølager , Harris, Pernille and Peters, Günther H.J. |