Structural studies of phycobiliproteins: a laboratory Biophysics teaching experiment

ONTIVEROS, MALLKU Q.; VIGIL MAXIMILIANO ANGEL; RINALDI, DEBORA E.; JUAN PABLO ROSSI; FERREIRA GOMES, MARIELA

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

Determining protein stability is essential for characterizing protein structure. There aremany traditional methods such as fluorescence, circular dichroism, hydrogen exchangemass spectrometry, differential scanning fluorescence, protein crystallization, and pulsedproteolysis to determine the structural stability of proteins and enzymes. Some of thesemethods are not readily available in biochemistry teaching experiments.Phycobiliproteins (PBPs) are fluorescent hydrophilic proteins characterized by multiplechromophores called phycobilins covalently linked through a cysteine residue to theirpolypeptide structures. PBPs are ideal for extensive protein folding exercises because thespectral properties of these chromophores are strongly influenced by the protein state.Based on this, we present a laboratory practice for determining PBPs stability bymonitoring urea-induced denaturation of a PBPs using absorbance and fluorescence.The PBPs used in this work was the phycocyanin (PC) isolated from the cyanobacteriumArthrospira, which was obtained from dietary supplement capsules or powder. Purificationof proteins (mainly PC) is fast and simple. Monitoring of PC denaturation by increasingconcentrations of urea was performed through the decrease of the absorption maximumat 625 nm and fluorescence maximum at 650 nm.Results show that the unfolding process of PC is reversible and can be explained by a twostatemodel. The experimentally determined Gibbs free energy upon unfolding in waterwas highly reproducible using both methods and correlated well to literature values.Finally, using this simple and inexpensive laboratory exercise, students can performcritical experiments to evaluate the unfolding process and study the protein structure.AcknowldegmentsWith grants from ANPCYT PICT 2014 #2014, PICT 2015 #0067 and PICT 2018 #00748, CONICET PIP 11220150100250CO and UBA 2018-2022: 20020170100081BA.*Both authors contributed equally to this work.