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XLIII RA ? SAB 2014 PROTEINS AND NUCLEIC ACIDS _____________________________________________________ 108 PNA-14] Preliminary Studies on the Folding Mechanism of Phenylalanine Hydroxylase Castro, I, Santos, J , Bredeston, L IQUIFIB (UBA-CONICET)/ Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina. Phenylalanine hydroxylase from Legionella pneumophila (LpPAH) catalyzes the hydroxylation of L-Phe to L-Tyr using tetrahydrobiopterin (BH4) and non-heme Fe2+ as cofactors, and oxygen as additional substrate with an optimum temperature at 45°C. 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 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.