Resonance Raman and vibrational mode analysis used to predict ligand geometry for docking simulations of a water soluble porphyrin and tubulin

Abstract

The ability to modify the conformation of a protein by controlled partial unfolding may have practical applications such as inhibiting its function or providing non-native photosensitive properties. A water-soluble porphyrin, meso-tetrakis (p-sulfonatophenyl) porphyrin (TSPP), non-covalently bound to tubulin can be used as a photosensitizer, which upon irradiation can lead to conformational changes of the protein. To fully understand the mechanism responsible for this partial unfolding and determine the amino acid residues and atoms involved, it is essential to find the most likely binding location and the configuration of the ligand and protein. Techniques typically used to analyze atomic position details, such as nuclear magnetic resonance and X-ray crystallography, require large concentrations, which are incompatible with the dilute conditions required in experiments for photoinduced mechanisms. Instead, we develop an atomistic description of the TSPP-tubulin complex using vibrational mode analysis from density functional theory calculations correlated to resonance Raman spectra of the porphyrin paired with docking simulations. Changes in the Raman peaks of the porphyrin molecule correlate with changes in its structural vibrational modes when bound to tubulin. The data allow us to construct the relative geometry of the porphyrin when bound to protein, which are then used with docking simulations to find the most likely configuration of the TSPP-tubulin complex.

Partial Unfolding of Tubulin Heterodimers Induced by Two-Photon Excitation of Bound meso-Tetrakis(sulfonatophenyl)porphyrin

“…The fact that the decrease of emission intensity from the Trp residues occurs without changes in the absorption of the same residues does suggest that the tubulin undergoes structural changes without chemical changes of the aromatic amino acid residues. A previous docking study suggested that the most likely binding configuration between TSPP and tubulin places one of the sulfonic acid groups of TSPP 6.75 Å away from Trp 346. This binding location is near the region in which tubulin heterodimers bind to each other for polymerization.…”

“…How can the data from Figure and Figure be reconciled? We have recently shown that binding to proteins distorts the porphyrin macrocycle and changes the group symmetry of the chromophore , from a D 2 h to a ruffled conformation which has no defined point group symmetry. This change may lift TPE symmetry restrictions and allow transitions between orbitals that are otherwise forbidden .…”

“…A previous docking 52 study suggested that the most likely binding configuration between TSPP and tubulin places one of the sulfonic acid groups of TSPP 6.75 Å away from Trp 346. This binding location is near the region in which tubulin heterodimers bind to each other for polymerization.…”

“…This change may lift TPE symmetry restrictions and allow transitions between orbitals that are otherwise forbidden. 24 Once TSPP binds tubulin, the change in symmetry forced by the docking 52 enables one or both of the following effects: the populated first excited singlet state is the same as for OPE and has the same decay lifetime, and/or from this state, intersystem crossing to the triplet state is restored to levels similar to the OPE. TPE fluorescence (Figure 10A) is identical upon irradiation with the 1 kHz or the 80 MHz sources because, even though the second source excites a fraction of TSPP to higher states, these do not contribute to the emission of the molecule or to populate the triplet state.…”