Calorimetría de titulación isotérmica

La unión reversible de un ligando a una proteína o ácido nucleico constituye, con la excepción de los procesos fotoquímicos, el modo universal de iniciación de los procesos biológicos que tienen lugar en los seres vivos (Gutfreund, 1995). El entendimiento de estos procesos de reconocimiento molecular es de fundamental importancia en la biología moderna. Su estudio consiste en determinar la estequiometría, la constante de equilibrio y la energética del proceso. La calorimetría de titulación isotérmica es la única técnica disponible capaz de evaluar a través de un solo experimento estos tres componentes sin ambigüedad. Casi cualquier reacción química o cambio físico producido en un sistema es acompañado por la liberación o absorción de calor. En este sentido los calorímetros pueden ser considerados como detectores universales y a la vez promiscuos, con las ventajas y desventajas que esto implica. A diferencia de los métodos ópticos, las medidas calorimétricas no requieren la incorporación de marcas específicas y se pueden realizar en sistemas opacos, turbios o heterogéneos (por ejemplo, células en suspensión), y bajo una amplia gama de condiciones biológicamente relevantes (temperatura, pH, fuerza iónica, etc.). El primer calorímetro isotérmico fue construido por Lavoiser y Laplace en 1780 (memoria sobre el calor). Desde ese momento se ha desarrollado una amplia variedad de instrumentos, pero no fue hasta 1990 cuando aparecieron en el mercado calorímetros sencillos y con una sensibilidad muy alta, que permitieron estudiar reacciones bioquímicas de asociación. Este tipo de calorímetro, donde se añade un ligando paso a paso a una solución de macromoléculas a temperatura constante y bajo agitación continua, se conoce como calorimetría de titulación isotérmica o ITC por su sigla en inglés (Isothermal Tritation Calorimetry).Facultad de Ciencias Exacta

Cooperativity in binding processes: New insights from phenomenological modeling

Cooperative binding is one of the most interesting and not fully understood phenomena involved in control and regulation of biological processes. Here we analyze the simplest phenomenological model that can account for cooperativity (i.e. ligand binding to a macromolecule with two binding sites) by generating equilibrium binding isotherms from deterministically simulated binding time courses. We show that the Hill coefficients determined for cooperative binding, provide a good measure of the Gibbs free energy of interaction among binding sites, and that their values are independent of the free energy of association for empty sites. We also conclude that although negative cooperativity and different classes of binding sites cannot be distinguished at equilibrium, they can be kinetically differentiated. This feature highlights the usefulness of pre-equilibrium time-resolved strategies to explore binding models as a key complement of equilibrium experiments. Furthermore, our analysis shows that under conditions of strong negative cooperativity, the existence of some binding sites can be overlooked, and experiments at very high ligand concentrations can be a valuable tool to unmask such sites.Instituto de Física de Líquidos y Sistemas BiológicosFacultad de Ciencias Exacta

Activation of Archaeoglobus fulgidus Cu+-ATPase CopA by cysteine

AbstractCopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across the cell membrane. Millimolar concentration of Cys dramatically increases (≅800%) the activity of CopA and other PIB-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (≅1 μM) together with the low Cu+–Cys KD (<10−10M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu+ chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2–20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu+, suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP Km suggesting no changes in the E1↔E2 equilibrium. Characterization of Cu+ transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu+ can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu+–Cys complex

Cooperativity in binding processes: New insights from phenomenological modeling

Cooperative binding is one of the most interesting and not fully understood phenomena involved in control and regulation of biological processes. Here we analyze the simplest phenomenological model that can account for cooperativity (i.e. ligand binding to a macromolecule with two binding sites) by generating equilibrium binding isotherms from deterministically simulated binding time courses. We show that the Hill coefficients determined for cooperative binding, provide a good measure of the Gibbs free energy of interaction among binding sites, and that their values are independent of the free energy of association for empty sites. We also conclude that although negative cooperativity and different classes of binding sites cannot be distinguished at equilibrium, they can be kinetically differentiated. This feature highlights the usefulness of pre-equilibrium time-resolved strategies to explore binding models as a key complement of equilibrium experiments. Furthermore, our analysis shows that under conditions of strong negative cooperativity, the existence of some binding sites can be overlooked, and experiments at very high ligand concentrations can be a valuable tool to unmask such sites.Instituto de Física de Líquidos y Sistemas BiológicosFacultad de Ciencias Exacta

A Two-Stage Model for Lipid Modulation of the Activity of Integral Membrane Proteins

Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes

Comparison between negative cooperativity and different sites in pre-equilibrium conditions.

<p>(A) Time course of site occupation for a macromolecule with two identical sites for negative cooperativity (continuous lines) and two classes of binding sites without interactions (dash-dotted lines) for the following initial ligand concentrations (μM): 300 (red), 100 (orange), 50 (green), 25 (turquoise), 10 (blue) and 5 (violet). Time courses simulations were obtained under the same conditions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146043#pone.0146043.g005" target="_blank">Fig 5</a>. (B) The difference Δ〈<i>n</i>〉 = 〈<i>n</i>〉_coop—〈<i>n</i>〉_{diff sites} was calculated for each ligand concentration and represented as a function of time. For clarity reasons only 6 representative time courses traces that gave rise to the equilibrium data points of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146043#pone.0146043.g005" target="_blank">Fig 5</a> are shown.</p

Dependence of the Hill coefficient on the interaction and association free energies.

<p>The Hill coefficient was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146043#pone.0146043.g003" target="_blank">Fig 3</a> from the binding isotherms obtained for identical sites with <i>K</i>_o = 1 (μM^-1) and different values of the cooperativity factor ω (main plot) and for identical sites with <i>K</i>_o varying from 0.01 to 100 (μM^-1) and a fixed value of the cooperativity factor ω = 8 (inset). <i>ΔG</i>^o_int (main plot) and <i>ΔG</i>^o_assoc (inset), were calculated using Eqs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146043#pone.0146043.e010" target="_blank">10</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146043#pone.0146043.e009" target="_blank">9</a> respectively. Continuous lines are the graphical representation of polynomial functions fitted to the simulated data.</p