Abstract

We report on the response of reaction center (RC) from Rhodobacter sphaeroides (an archetype of membraneproteins) to the exposure at high temperature. The RCs have been solubilized in aqueous solution of thedetergent N,N-dimethyldodecylamine-N-oxide (LDAO). Changes in the protein conformation have beenprobed by monitoring the variation in the absorbance of the bacteriochlorine cofactors and modification inthe efficiency of energy transfer from tryptophans to cofactors and among the cofactors (throughfluorescence measurements). The RC aggregation taking place at high temperature has been investigated bymeans of dynamic light scattering. Two experimental protocols have been used: (i) isothermal kinetics, inwhich the time evolution of RC after a sudden increase of the temperature is probed, and (ii) T-scans, inwhich the RCs are heated at constant rate. The analysis of the results coming from both the experimentsindicates that the minimal kinetic scheme requires an equilibrium step and an irreversible process. Theirreversible step is characterized by a activation energy of 205±14 kJ/mol and is independent from thedetergent concentration. Since the temperature dependence of the aggregation rate was found to obey to thesame law, the aggregation process is unfolding-limited. On the other hand, the equilibrium process betweenthe native and a partially unfolded conformations was found to be strongly dependent on the detergentconcentration. Increasing the LDAO content from 0.025 to 0.5 wt.% decreases the melting temperature from49 to 42 °C. This corresponds to a sizeable (22 kJ/mol at 25 °C) destabilization of the native conformationinduced by the detergent. The nature of the aggregates formed by the denatured RCs depends on thetemperature. For temperature below 60 °C compact aggregates are formed while at 60 °C the clusters are lessdense with a scaling relation between mass and size close to that expected for diffusion-limited aggregation.The aggregate final sizes formed at different temperatures indicate the presence of an even number ofproteins suggesting that these clusters are formed by aggregation of dimers.

Autore Pugliese

• Mallardi Antonia

Tutti gli autori

• Palazzo G.; Lopez F.; Mallardi A.

Titolo volume/Rivista

Biochimica et biophysica acta. Proteins and proteomics

Anno di pubblicazione

2010

ISSN

1570-9639

ISBN

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Numero di citazioni Wos

Nessuna citazione

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Numero di citazioni Scopus

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Settori ERC

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Codici ASJC

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