Coupling of Protonation, Reduction, and Conformational Change in azurin from Pseudomonas aeruginosa Investigated with Free Energy Measures of Cooperativity

Abstract

We used free energy calculations within a continuum electrostatics model to analyze the coupling of protonation, reduction, and conformational change in azurin from Pseudomonas aeruginosa (PaAz). PaAz was characterized extensively with a variety of experimental methods. Experimentally determined pK a values and pH-dependent reduction potentials are used to validate our computational model. It is well-known from experiment that the reduction of the copper center is coupled to the protonation of at least two titratable residues (His-35 and His-83) and to the flip of the peptide bond between Pro-36 and Gly-37. Free energy measures of cooperativity are used for a detailed analysis of the coupling between protonation, reduction, and conformational change in PaAz. The reduction of the copper center, the protonation of His-35, and peptide flip are shown to be cooperative. Our results show that cooperativity free energies are useful in detecting and quantifying thermodynamic coupling between events in biomolecular systems. The protonation of His-35 and the peptide flip are found to be so tightly coupled that these events happen effectively concerted. This concerted change results in a marked alteration of the electrostatic surface potential of azurin that might affect the interaction of azurin with its binding partners.

Converting the Yeast Arginine Can1 Permease to a Lysine Permease

“…The pH-dependent protonation probability of all titratable protein residues in selected protein-ligand complexes was calculated by a continuum electrostatics/Monte Carlo approach (41)(42)(43).…”

Thermodynamics of Transport Through the Ammonium Transporter Amt-1 Investigated with Free Energy Calculations

“…Four alternative hydrogen positions for protonated forms of carboxylic acids were added to represent the syn and trans configurations of the dissociable proton at each carboxyl oxygen atom. The corresponding angles and bond lengths were taken from ref 69.…”

On the interaction of two different types of ligands binding to the same molecule part I: basics and the transfer of the decoupled sites representation to systems with n and one binding sites

“…This problem is of biophysical interest as proteins involved in electron transfer are often located in membranes with pH-gradient. Moreover, it can be applied to any other transporter molecule with proton binding sites or receptors with different types of ligands which are frequently a subject of investigation [3,8,9,13,18,19]. Also from a mathematical point of view this transfer is not trivial as one has to deal with polynomials of the polynomial ring in two variables C [ , κ].…”