title: Metastability of papain and the molecular mechanism for its sequential acid-denaturation.
abstract: Acid unfolding of non-inhibited papain at pH 2 was studied by means of spectroscopic and electrophoresis techniques as well as activity assays. We found a molten globule like species (A state) similar to that previously reported for bromelain and S-carboxy-methyl-papain. We demonstrated that this A state is not thermodynamically stable but a metastable conformer which decays into an unfolded conformation in a few hours. The mechanism of acid unfolding to the A state proved to be completely irreversible, with a biphasic time evolution of spectroscopic signals characteristic of the existence of a kinetic intermediate. This latter species showed properties in-between native and A state such as secondary structure, exposition of hydrophobic area and tryptophan environment, but a native like hydrodynamic radius. Native papain seems to unfold at acid pH through at least two kinetic barriers, being its pro-region mandatory to conduct and stabilize its active structure. Computer simulations of acid unfolding, followed by ANS docking, identified three regions of cavity formation induced by acid media which might be used as regions to be fortified by protein engineering in the quest for extreme-resistant proteases or as hot-spots for protease inactivation.
authors.affiliation: Universidad Autónoma de San Luis Potosí, Coordinación Académica Región Altiplano, COARA, Carretera a Cedral Km 5+600, Ejido San José de las Trojes, 78700 Matehuala, San Luis Potosí, Mexico.
authors.forename: Rosa Eréndira
authors.initials: RE
authors.lastname: Fosado-Quiroz
authors.forename: Arturo
authors.initials: A
authors.lastname: Rojo-Domínguez
mesh_terms: D000143:Acids / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010206:Papain / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D013816:Thermodynamics
chemical_list: D000143:Acids; D010206:Papain