Browsing by Author "Baez, Mauricio"

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    Amperometric and Voltammetric Determination of Oxytetracycline in Trout Salmonid Muscle Using Multi-wall Carbon Nanotube, Ionic Liquid and Gold Nanoparticle Film Electrodes
    (2012) Nagles Vidal, Edgar Orlando; Alvarez, Paola; Arancibia Moya, Verónica; Baez, Mauricio; Garretón, Virginia; Ehrenfeld Stolzenbach, Katia Nicole
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    Effect of temperature and nucleotide on the binding of BiP chaperone to a protein substrate
    (2023) Rivera, Maira; Burgos-Bravo, Francesca; Engelberger, Felipe; Asor, Roi; Lagos-Espinoza, Miguel I. A.; Figueroa, Maximiliano; Kukura, Philipp; Ramirez-Sarmiento, Cesar A.; Baez, Mauricio; Smith, Steven B.; Wilson, Christian A. M.
    BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding. Recent structural studies have provided new insights into BiP's allostery; however, the influence of temperature on the coupling between substrate and nucleotide binding to BiP remains unexplored. Here, we study BiP's binding to its substrate at the single molecule level using thermo-regulated optical tweezers which allows us to mechanically unfold the client protein and explore the effect of temperature and different nucleotides on BiP binding. Our results confirm that the affinity of BiP for its protein substrate relies on nucleotide binding, by mainly regulating the binding kinetics between BiP and its substrate. Interestingly, our findings also showed that the apparent affinity of BiP for its protein substrate in the presence of nucleotides remains invariable over a wide range of temperatures, suggesting that BiP may interact with its client proteins with similar affinities even when the temperature is not optimal. Thus, BiP could play a role as a "thermal buffer" in proteostasis.
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    Temperature dependent mechanical unfolding and refolding of a protein studied by thermo-regulated optical tweezers
    (2023) Rivera, Maira; Mjaavatten, Are; Smith, Steven B.; Baez, Mauricio; Wilson, Christian A. M.
    Temperature is a useful system variable to gather kinetic and thermodynamic information from proteins. Usually, free energy and the associated entropic and enthalpic contributions are obtained by quantifying the conformational equilibrium based on melting experiments performed in bulk conditions. Such experiments are suitable only for those small single-domain proteins whose side reactions of irreversible aggregation are unlikely to occur. Here, we avoid aggregation by pulling single-protein molecules in a thermo-regulated optical tweezers. Thus, we are able to explore the temperature dependence of the thermodynamic and kinetic pa-rameters of MJ0366 from Methanocaldococcus jannaschii at the single-molecule level. By performing force-ramp experiments be-tween 2 degrees C and 40 degrees C, we found that MJ0366 has a nonlinear dependence of free energy with temperature and a specific heat change of 2.3 5 1.2 kcal/mol*K. These thermodynamic parameters are compatible with a two-state unfolding/refolding mechanism for MJ0366. However, the kinetics measured as a function of the temperature show a complex behavior, suggesting a three-state folding mechanism comprising a high-energy intermediate state. The combination of two perturbations, temperature and force, reveals a high-energy species in the folding mechanism of MJ0366 not detected in force-ramp experiments at constant temperature.