16 research outputs found
Stabilization of Tryptophan Hydroxylase 2 by L-Phenylalanine Induced Dimerization
Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rateâlimiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Fullâlength 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, NÎ47ârch 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 14â and 11âfold higher halfâlives compared to rch TPH2 and NÎ47ârch 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 NÎ47ârch TPH2 are found to shift from polyphasic to apparent twoâstate by the addition of lâTrp or lâPhe. Analytical gel filtration revealed that rch TPH2 and NÎ47ârch TPH2 reside in a monomerâdimer equilibrium which is significantly shifted toward dimer in the presence of lâPhe. The dimerizing effect induced by lâPhe is accompanied by a stabilizing effect, which resulted in a threefold increase in halfâlives of rch TPH2 and NÎ47ârch TPH2. Addition of lâPhe to the purification buffer significantly increases the purification yields, which will facilitate characterization of hTPH2