Investigating the binding-release mechanism of periplasmic ferric binding protein by ph variations and point mutations

Official URL: http://risc01.sabanciuniv.edu/record=b1534431 (Table of Contents)

Differences between the conformations of the ligand-bound and unbound forms of proteins provide clues on deciphering residues that have a direct effect on binding mechanisms. Using molecular dynamic (MD) simulations as a basis, conformational changes that take place on time scales much slower than those accessible by MD may be investigated by supplementary methods such as perturbation response scanning (PRS). Since proteins are complex macromolecules, to sample their conformational energy landscapes we applied both global and local perturbations in different combinations. Global perturbations are related with environmental changes such as (i) different values of ionization strength of the solution which mimic the various salt environments experienced by the protein in vivo, (ii) or protonation of a group of residues to mimic a different pH environment. Local perturbations are related to specific point perturbations on protein such as, (i) protonation or (ii) mutation of a single residue to locate the points that controls a conformational change in proteins. In this work, the apo and holo (Fe3+ bound) forms of the periplasmic ferric binding protein systems (hFBP) of the gram negative bacteria haemophilus influenzae was selected as the model system. PRS studies showed us that D52 and D47 residues are the ones that give the highest value for the fractional contribution of the eigenvalue of the response matrix. Therewithal, using pKa calculations, a particular charged residue (D52) (out of a total of 98) was found to be the most sensitive to subtle pH variations in the physiological range. The effect of single point protonation (D52+) and mutation (D52A) is investigated via a series of MD simulations. The effect of IS (0.15 mM and ~0 mM) and pH (5 and 7.4 ) change is also studied to monitor the conformations sampled. For apo FBP, the kinetics of synergistic anion binding and release was monitored and was consistently manipulated by varying these conditions, while no apparent conformational change was observed in the protein. For holo FBP, protonated and Ala mutants of D52 consistently trigger opening of the iron binding site in an ensemble of simulations, while elevated IS consistently traps the closed forms. We categorized our series of MD trajectories as open, partially open, and closed due to coordination level and mapping of iron ion inside the active site. Our results lend clues as to how the environment versus single residue perturbations may be utilized for regulation of binding modes in hFBP systems.