XPLORING THE FOLDING MECHANISM OF A THERMOPHILIC alpha–HELICAL MEMBRANE PROTEIN WITH THE USE OF SDS

ROMAN, ERNESTO A.; RECOULAT ANGELINI, ALVARO A.; GONZALEZ FLECHA, F LUIS; JOSÉ M ARGUELLO

Aguas de Lindoia

Congreso; 51th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology (SBBq) 46th Congress of Brazilian Biophysical Society (SBBf)/ Lafebs; 2022

Brazilian Biophysical Society

The practical difficulty to produce and purify membrane proteins, in addition to the complex interaction with the surrounding lipids or detergents, is the main reason for which folding mechanism studies are focused almost exclusively for soluble proteinsa. Furthermore, α-helical membrane proteins are typically resistant to classical denaturation agents like urea and guanidinium hydrochloride, but are susceptible to ionic detergents, like sodium dodecyl sulfate (SDS)b. In this work, we explore the SDS-induced denaturation of AfCopA, a thermophilic α-helical membrane protein from Arhcaeglobus fulgidus. For this, the protein was obtained as described previouslyc, and reconstituted in mixed micelles composed of phoshoplipids and a nonionic detergent. Results shows that the addition of SDS disrupts the structure of the protein, loosing it’s catalytic activity and showing a decrease on the intrinsic fluorescence and the signal of a fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid, bound to the protein. However, Far UV circular dichroism spectroscopy results show that the interaction doesn’t alter the secondary structure of the protein. By changing the initial concentration of amphiphiles, we demonstrated that the effect on the signal are dependent on the molar fraction of SDS present on the mixed micelles. The observed transitions were proven reversible by the dilution of the detergent, which allowed us to perform a thermodynamic analysis of the process. Exploring the denaturation process trough multiple probes allow us to postulate a "mechanism” by which this protein re-folds and recovers its catalytic activity, described by three successive stages in the renaturation process.