CONICET | Buscador de Institutos y Recursos Humanos

INTRODUCTION Membrane proteins from hyperthermophilic organisms are particularly interesting because of their extraordinary stability, however, little is known about the factors that determine their stability. In this work we attempt to characterize the kinetics of the spontaneous inactivation of CopA, a thermophilic membrane protein from Archaeoglobus fulgidus. This enzyme retains its thermophilic characteristics when it is expressed in E. coli with maximum activity at 75ºC and Ea =103 kJ/mol [1]. METHODS CopA was heterologously expressed in E. coli, solubilized in dodecylmaltoside (DDM), and Ni+-affinity purified [1]. Cu+-ATPase activity was evaluated by measuring the release of Pi from ATP at 75ºC. The enzyme thermal inactivation was assayed by incubating CopA at different temperatures and measuring its ATPase activity at 75ºC on aliquots taken at different times. Three Cop A preparations were assayed following this procedure: (A) E coli membranes obtained after CopA induction (B) purified protein, and (C) CopA reconstituted in asolectin/DDM mixed micelles. The inactivation was also monitored by Tris/Tricine SDS/PAGE and fluorescence spectroscopy. RESULTS AND DISCUSION Thermal stability of CopA was compared among the different enzyme preparations. In all the conditions the enzyme activity showed an irreversible exponential decrease. As it was expected CopA inactivates much slower in E coli membranes and faster in the DDM solubilized preparation. The kinetic coefficients of thermal inactivation at 75°C were: ki(A) = 0.039 ± 0.02 min-1, ki(B) = 0.33 ± 0.06 min-1, ki(C) = 0.054 ± 0.01 min-1. The dependence of the inactivation kinetics on the incubation temperature was analyzed according to the Arrenhius equation. The obtained activation energies were Ea(A) =185 ± 20 kJ/mol, Ea(B) = 219 ± 12 kJ/mol, Ea(C) =186 ± 15 kJ/mol, being the differences in the pre-exponetial factor. Interestingly, the Ea values are not significantly different and they are in the same order of those corresponding to mesophilic P-ATPases [2]. Native and inactivated Cop A migrated as a single band after SDS/PAGE indicating that the inactivation was not associated with either fragmentation or formation of SDS-stableaggregates of the protein. We also determined the binding of ANS to the purified protein as a function of the incubation time at 75°C and found that ANS fluorescence intensity irreversibly decay to a constant value, which is the 70% of the initial fluorescence intensity, following a first order kinetics with k(B)= 0.031±0.002 min-1.CONCLUSION The results presented in this work show that thermophilic membrane proteins are more stable than their mesophilic counterparts in similar conditions [2]. The first-order rate of thermal inactivation suggests a two-state process involving only fully active and inactive molecules and the similarity between the kinetics for inactivation and binding of ANS suggest that inactivation is the result of a conformational transition that, given the high value of the remaining ANS fluorescence, still conserves large hydrophobic regions. Supported by NSF Grants MCM-0235165 and OISE-0436435 to JMA and UBACyT B089, CONICET, and ANPCyT PICT 11138 to LGF REFERENCES (1) Mandal, A. K., Cheung, W. D., and Argüello, J. M. (2002) J Biol Chem 277, 7201-7208 (2) Levi V, Rossi JPFC, Echarte MM, Castello PR, González Flecha FL. (2000). J Membrane Biol 173:215-225

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