Combination of Resonance Raman Spectroscopy and Docking Simulations to Study the Nonspecific Binding of a Free-Base Porphyrin to a Globular Protein

Abstract

Understanding the conformational changes induced by small ligands noncovalently bound to proteins is a central problem in biophysics. We focus on the binding location of the water-soluble porphyrin, meso-tetrakis (p-sulfonatophenyl) porphyrin, to a globular protein, β-lactoglobulin, which has been observed to partially unfold when irradiated by laser light. Identifying the binding location is necessary to determine the mechanism of action as well as the atoms and residues involved in the photoinduced partial unfolding. Such atomic details are typically investigated by nuclear magnetic resonance or X-ray crystallography. However, for biomolecules in solution at the low concentrations (μM) required to deliver uniform laser irradiation, these traditional techniques do not currently provide sufficient information, and one must rely upon less direct spectroscopic methods. We describe a method that uses resonance Raman spectroscopy and density functional theory (DFT) to select the most likely binding configuration among a set of solutions yielded by computational docking algorithms. This methodology may be generalized to use with other ligand-protein complexes where the ligand structure is amenable to DFT simulations.

Photoinduced partial unfolding of tubulin bound to meso‐tetrakis(sulfonatophenyl) porphyrin leads to inhibition of microtubule formation in vitro

“…What was the initial biomedical investigation, has now morphed into a more basic biophysical approach to address the questions raised by the effects of laser irradiation on proteins. For instance, using a combination of Resonance Raman Spectroscopy (RRS), density functional theory (DFT) and docking simulations we have started to address the problem of establishing the location of the ligand [9]. We demonstrated that the approach is promising by establishing that certain porphyrins are able to prompt conformational changes in small globular proteins [7,8].…”

Combined Use of Optical Spectroscopy and Computational Methods to Study the Binding and the Photoinduced Conformational Modification of Proteins When NMR and X-Ray Structural Determinations Are Not an Option

“…Following is a description of a recent methodology developed in our research group that uses a combination of experimental and computational techniques in order to obtain more detailed spatial information regarding the ligand/ protein complex. With this particular approach, one combines RRS with density functional theory (DFT) calculations and computational docking simulations (Parker, Thomas, Morisson, & Brancaleon, 2012).…”

“…This approach makes use of the 6-31G * basis set for the atoms in the molecule as long as the details of meso-or the beta-substituents are either ignored or replaced with a pseudo-atom that represents the characteristics of that substituents. For instance in one of our studies, the sulfonic acid groups (SO 3 À ) at the terminus of each phenyl in TSPP and replacing them with a hydrogen atom, that is, the simulation was reduced to a molecule of tetraphenylporphyrin (TPP) involving only hydrogen, carbon, and nitrogen atoms (Parker et al, 2012). Such simplification is justified by the fact that the laser wavelength (413 nm) primarily excites the porphyrin macrocycle; and therefore, the vibrational modes of the macrocycle will dominate the RR spectra which will minimize the effects of the groups appended to the phenyl meso-substituents.…”

“…This impasse can be resolved by dividing the space into smaller but overlapping regions of space that sample both the inside and the surface of the protein (Parker et al, 2012). The overlap region is somewhat arbitrary, but it has to be larger than at least 60% of the volume of the ligand in order to ensure that a docking site in a region of the protein is still represented in the next region.…”

“…(4.52), under the assumption that I k 0 is the same for a and b, and o k 0 is the frequency for modes a and b. In the case of the TSPP model, only 33 modes are recorded (Parker et al, 2012) in RRS ( Fig. 4.9), and only the lowest six vibrational orders are necessary to determine the distortions.…”