Structural prediction of a rhodamine-based biosensor and comparison with biophysical data

Abstract

The predicted structure has been calculated for a protein-based biosensor for inorganic phosphate (Pi), previously developed by some of us (Okoh et al., Biochemistry, 2006, 45, 14764). This is the phosphate binding protein from Escherichia coli labelled with two rhodamine fluorophores. Classical molecular dynamics and hybrid Car-Parrinello/molecular mechanics simulations allow us to provide molecular models of the biosensor both in the presence and in the absence of Pi. In the latter case, the rhodamine fluorophores maintain a stacked conformation in a 'face A to face B' orientation, which is different from the 'face A to face A' stacked orientation of free fluorophores in aqueous solution (Ilich et al., Spectrochim. Acta, Part A, 1996, 52, 1323). A protein conformation change upon binding Pi prevents significant stacking of the two rhodamines. In both states, the rhodamine fluorophores form hydrophobic contact with LEU291, without establishing significant hydrogen bonds with the protein. The accuracy of the models is established by a comparison between calculated and experimental absorption and circular dichroism spectra.

Development of a Reagentless Biosensor for Inorganic Phosphate, Applicable over a Wide Concentration Range

“…Mutations to the cleft surface resulted in small or no signal change upon P i addition, so the affinity could not be readily determined. Although the predicted structure of the rhodamines in the apo structure suggests little contact with surface amino acids, 21 this aspect is little understood and mutations in the cleft might affect indirectly the structural changes that disrupt the rhodamine interaction. In other words, mutations that weaken the interaction across the cleft may mean that cleft closure becomes unfavored thermodynamically.…”

Absorption and Emission Spectra of a Flexible Dye in Solution: A Computational Time-Dependent Approach

“…32,33 Several applications in recent years confirm the reliability and robustness of the GLOB and other related approaches. [34][35][36][37][38][39][40][41][42] Although the classical MD step can be performed also employing conventional periodic conditions in order to use standard codes (here GROMACS), NPBCs are more suitable for QM computations based on localized basis sets. Then the extraction of representative frames can be carried out following different schemes.…”

“…Implicit solvation methods, such as the Polarizable Continuum Model (PCM), offer the undeniable advantage of correctly describing the average polarization of the environment and its effect on the chromophore without any explicit sampling of solvent coordinates, but they are, of course, unable to capture specific solvent effects, especially in the cybotactic region. , This has led to the proposal of an integrated discrete/continuum model able to describe both short-range and bulk solvent effects in a fully dynamical framework. The so-called GLOB model enforces nonperiodic boundary conditions (NPBC) around a rather large cluster of explicit solvent molecules by means of both PCM reaction field and specific short-range potentials minimizing boundary effects. , Several applications in recent years confirm the reliability and robustness of the GLOB and other related approaches. − Although the classical MD step can be performed also employing conventional periodic conditions in order to use standard codes (here GROMACS), NPBCs are more suitable for QM computations based on localized basis sets. Then the extraction of representative frames can be carried out following different schemes.…”

Chapter 9. First Principles Methods in Biology: From Continuum Models to Hybrid Ab initio Quantum Mechanics/Molecular Mechanics

“…As yet, most QM/MM investigations on optical chromophore properties have been focused on solvent effects on absorption spectra, [242][243][244] whereas studies on biomolecular environmental shifts on optical spectra are still relatively rare. [245][246][247][248][249][250][251][252] That is, the full conformational flexibility of large protein environments at ambient temperature based on MD and its influence on absorption or emission band positions and shapes, e.g. inhomogeneous broadening of spectra, is only rudimentary investigated.…”