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

Abstract

The irradiation of the complex formed by meso-tetrakis (sulfonatophenyl) porphyrin (TSPP) and tubulin was investigated as well as its effects on the structure and function of the protein. We have used tubulin as a model target to investigate whether photoactive ligands docked to the protein can affect the structure and function of the protein upon exposure to visible light. We observed that laser irradiation prompts bleaching of the porphyrin which is accompanied by a sharp decrease (∼2 ns) in the average fluorescence lifetime of the protein and a change in the dichroic spectrum consistent with a decrease of helical structure. The result indicated the photoinduced partial unfolding of tubulin. We also observed that such partial conformational change inhibits the formation of microtubules in vitro. We investigated whether photosensitization of reactive oxygen species was responsible for these effects. Even upon removal of O2 the protein still undergoes conformational changes indicating that irradiation of the bound porphyrin does not require the presence of O2 to prompt conformational and functional effects opening the possibility that other mechanisms (e.g., charge transfer) are responsible for the photoinduced mechanism.

Effects of Visible‐Light Irradiation of Protoporphyrin IX on the Self‐Assembly of Tubulin Heterodimers

“…Changes in the emission lifetime of Trp residues can be correlated with modifications of their immediate environment [20] . The experiments show that the quenching of the emission of tubulin is accompanied by a decrease of ~ 0.3 ns in its average fluorescence lifetime (Figure 9), similar to that observed in other porphyrin/tubulin complexes [13] . Closer inspection shows that the major effect of the irradiation appears to be on the duration of the longer-lived component (Table 2).…”

“…It had been previously observed [13, 17] that addition of porphyrins to a tubulin aqueous solution decreases its intrinsic fluorescence and produces a blue-shift of its emission maximum from 330 to 326 nm due to quenching of its more exposed Trp residues [13, 17] . Unlike those previous studies, where protein fluorescence was collected with λ ex = 280 nm (which prompts emission of both Trp and Tyr residues), in the current investigation experiments were conducted with λ ex = 294 nm where the Trp residues are selectively excited.…”

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

“…In addition, the proximity to the Trp 346 is consistent with the TSPP-induced quenching of tubulin fluorescence observed previously. (McMicken, 2014) The interior site ( Figure 5 (right)) yields a free energy of binding equal to -9.34 kcal/mol, thus comparable to the one of the exterior site. At this location TSPP is nestled between the α and β monomers.…”

“…However, we demonstrated that irradiation of the TSPP-tubulin complex inhibits formation of MT. (McMicken, 2014) We would, therefore, expect TSPP to bind at a location where the photoinduced partial unfolding change may interfere with the self-assembly of MT.…”

“…(McMicken, 2014) Tubulin heterodimers are ubiquitous in eukaryotic cells and consist of two globular proteins both 55 kDa in mass. (Dutcher, 2001) Tubulin heterodimers are the building blocks of MT which form the cytoskeleton of cells and are responsible for mitosis and other vital cellular functions.…”