Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase.
Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.
Iida
Tsukimi
T
Department of Food and Nutrition, Tsu City College, Mie, Japan.
Nishimura
Shigenori
S
Mochizuki
Maki
M
Uchiyama
Susumu
S
Ohkubo
Tadayasu
T
Urade
Yoshihiro
Y
Tanaka
Akiyoshi
A
Inui
Takashi
T
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006358:Hot Temperature; D019746:Intramolecular Oxidoreductases / Q000737:chemistry*; D054834:Lipocalins / Q000737:chemistry*; D051379:Mice; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D054834:Lipocalins; D011994:Recombinant Proteins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase