Spectral analysis of SDS-induced reversible denaturation of AfCopA, an alfa;-helical membrane protein

RECOULAT ANGELINI, ALVARO A.; GONZALEZ FLECHA, F LUIS

Congreso; XLIX Reunión Anual de la Sociedad Argentina de Biofísica; 2021

Sociedad Argentina de Biofísica

Membrane proteins play a central role in life by helping cells to communicate with their surroundings. They represent approximately one third of the coding proteins in all genomes, and alterations in their folding could lead to a number of known pathologies [1]. However, when comparing with soluble proteins, few studies are focused on their folding and stability due to practical difficulties [2]. Furthermore, α-helical membrane proteins are typically resistant to classical denaturation agents like urea and GnHCl, but are susceptible to ionic detergents, like sodium dodecyl sulfate (SDS) [3]. In this work, we explore the SDS-induced denaturation of AfCopA, a thermophilic α-helical membrane protein from Arhcaeglobus fulgidus, by analyzing the effect on the intrinsic fluorescence and the signal of a fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), bound to the protein. The intensity of both signals decrease by the addition of SDS to the protein, which is initially reconstituted in mixed micelles composed of phoshoplipids and a nonionic detergent. 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. Further analysis were conducted by analyzing the effect of SDS on the spectrum´s shape. Changes were subtle, but by using the spectral phasor approach [4], we could identify new transitions and obtain additional information about intermediate species in the folding mechanism.