IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Preliminary Studies on the Folding Mechanism of Phenylalanine Hydroxylase.
Autor/es:
CASTRO I; SANTOS J; BREDESTON L
Reunión:
Congreso; Reunión anueal de SAB; 2014
Institución organizadora:
Sociedad Argentína de Biofísica
Resumen:
Preliminary Studies on the Folding Mechanism of Phenylalanine Hydroxylase
Castro I, Santos J and Bredeston L
IQUIFIB (UBA-CONICET)/ Departamento de Química Biológica, Facultad de Farmacia y
Bioquímica, Universidad de Buenos Aires, Argentina. ignacas22@gmail.com
Phenylalanine hydroxylase from Legionella pneumophila (LpPAH) catalyzes the hydroxylation of
L-Phe to L-Tyr using tetrahydrobiopterin (BH4) and non-heme Fe2+
additional substrate with an optimum temperature at 45oC. LpPAH is a 272-residue protein which
has a mixed α/β topology that forms a dimer characterized by a large interface. Noteworthy, this
protein showed high thermostability, with a reported Tm of 79 °C (1) suggesting that the interface
between monomers may be the source of the high stability observed. Here, we have ask how
coupled the global stability of the dimer and monomers are. The protein was purified from E. coli
(35mg/mL) and its conformation was studied. Far-UV CD was compatible with the expected native
LpPAH secondary structure. Near-UV CD exhibits signatures of asymmetric environments for for
Trp and Phe residues, suggesting a rigid tertiary/quaternary structure. Unfolding followed by Trp
fluorescence was carried out. We detected an intermediate (Ieq) state that exhibits a significant
decrease in Trp fluorescence intensity by comparison with the native state, whereas Trp residues
seem to be in average partially exposed to the solvent as judged by the center of mass of emission
spectra and by comparison to the unfolded LpPAH. Work on the hydrodynamic behavior of LpPAH
by SEC-FPLC and light scattering is under progress to determine whether the first transition in the
equilibrium unfolding includes loss of quaternary structure. With financial support of UBA, CONICET
and ANPCyT.
1- Flydal MI et al. PLoS One. 2012;7(9):e46209.