Calorimetric analysis of the interactions between SDS and the catalytic domain of a thermophylic Cu(I) transport ATPase

GONZÁLEZ FLECHA, F. LUIS; RECOULAT ANGELINI, ALVARO A.; OLIVEIRA, CRISTIANO

Águas de Lindóia, SP

Congreso; 48th Annual Meeting of the Brazilian Society of Biochemistry and Molecular Biology; 2019

Brazilian Society of Biochemistry and Molecular Biology

Introduction: Although membrane proteins constitute about a third of the proteinsencoded in known genomes, folding and stability studies on these proteins havebeen impaired mainly due to experimental limitations. Helical membrane proteins areusually resistant to chemical denaturation by urea or guanidine hydrochloride.However, ionic detergents, such as Sodium Dodecyl Sulfate (SDS), have beenefficient denaturant for some of these a.Objectives: Here, the interaction between SDS and the catalytic domain of A. fulgidusCu(I) transport ATPase, an hiperthermophilic integral membrane protein will beexplored by Isothermal titatrion calorimetry (ITC). Previous results using fluorescencetechniques show that this interaction is reversible, with the presence of at least oneintermediary in the unfolding process.Matherials and methods: The protein was obtained as described previously b. ITCexperiments were performed using the metodology descrided by Sun et al c.Results: A multi-step binding interaction was detected. All the transitions shifted tohigher SDS concentrations when the protein concentration was incremented. Theformation of protein-surfactant complexes occurs below the Critical MiceleConcentration. Dialysis of the protein to eliminate the SDS recover the biologicalactivity, demostrating that the transitions are reversible.Discusion: An initial high affinity interaction occurs at a 3:1 SDS:Protein molar ratio.The thermogram shows more of 10 relevant transitions, to reach a final stoichiometryof 110 moles of SDS per mole of protein. Most of these transitions doesnt appear inthe fluorescence experiments. The next step will be to obtain structural information ofthese SDS-protein complexes using SAXS.Acknowledgements:This work was supported by Universidad de Buenos Aires(UBACYT 0306) and ANPCYT (PYCT 2016-4224)Keywords: Membrane Proteins, SDS, binding, folding.Referencesa- Roman EA, González Flecha FL (2014) Biomolecules, 4,354-373b- Tsuda T, Toyoshima C. (2009). EMBO J. 28,1782-1791.c- Sun Y et al (2015). Soft Matter, 11,7769-.