Binding of Porphyrins to Tubulin Heterodimers

Tian, Fang; Johnson, Eric; Zamarripa, Miguel; Sansone, Samuel; Brancaleon, Lorenzo

doi:10.1021/bm700687x

Cited by 25 publications

(55 citation statements)

References 70 publications

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“…This is consistent with non-covalent complexation between the dye and the protein through electrostatic interactions between one or more of the four negatively charged SO À 3 groups in TSPP and positively charged residues in the tubulin dimer [25] with the addition of the possible rotation of the phenyl rings in-plane with the porphyrins macrocycle. Conversely, the absorption of tubulin does not appear to change upon binding of TSPP.…”

Section: Absorptionsupporting

confidence: 76%

“…We confirmed that addition of tubulin to TSPP results in a $7 nm red shift associated with hypochromicity of the peak of the porphryin ( Figure 1A) [25]. This is consistent with non-covalent complexation between the dye and the protein through electrostatic interactions between one or more of the four negatively charged SO À 3 groups in TSPP and positively charged residues in the tubulin dimer [25] with the addition of the possible rotation of the phenyl rings in-plane with the porphyrins macrocycle.…”

Section: Absorptionsupporting

confidence: 72%

“…Spectra of tubulin were collected between 300 nm and 450 nm with excitation wavelength, l ex , at 295 nm. At this wavelength the 8 Trp residues of the tubulin dimer are exclusively excited over the other aromatic amino acids [25]. Spectra of TSPP were recorded in the 580-750 nm range using l ex ¼ 413 nm.…”

Section: Steady State Fluorescence Spectroscopymentioning

confidence: 99%

“…Irradiation of the TSPP/tubulin complex solution was carried out using a solid state 185 mW laser at 405 nm (Power Technology inc., Alexander, AR), which excites TSPP in the Soret band [25]. The laser beam is attenuated through a series of neutral density filters and defocused using a long focal length lens to make the irradiance delivered to the sample approximately 15 mW/cm 2 .…”

Section: Irradiationmentioning

confidence: 99%

“…, and that the binding does not interfere with the ability to form MT [25] so that any influence on tubulin self-assembly can be attributed to photoinduced effects. In addition, Guo et al showed that irradiation of rhodamine-tags triggers depolymerization of MT upon laser exposure [26], thus indicating that visible light could be used to affect the formation of MT.…”

mentioning

confidence: 99%

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

McMicken

Thomas

Brancaleon

2013

Journal of Biophotonics

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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.

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Section: Absorptionsupporting

confidence: 76%

Section: Absorptionsupporting

confidence: 72%

Section: Steady State Fluorescence Spectroscopymentioning

confidence: 99%

Section: Irradiationmentioning

confidence: 99%

mentioning

confidence: 99%

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

McMicken

Thomas

Brancaleon

2013

Journal of Biophotonics

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

et al. 2016

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The formation and the effects of the laser irradiation of the complex formed by protoporphyrin IX (PPIX) and tubulin was investigated. We have used tubulin as a model protein to investigate whether docked photoactive ligands can affect the structure and function of polypeptides upon exposure to visible light. We observed that laser irradiation in the Soret band prompts bleaching of the PPIX which is accompanied by a sharp decrease in the intensity and average fluorescence lifetime of the protein (dominated by the four tryptophan residues of the tubulin monomer). The kinetics indicate non-trivial effects and suggest that the photosensitization of the PPIX bound to tubulin prompts structural alterations of the protein. These modifications were also observed through changes in the energy transfer between Trp residues and PPIX. The result suggest that laser irradiation produces localized partial unfolding of tubulin and that the changes prompt modification of the formation of microtubules in vitro. Measurements of singlet oxygen formation were inconclusive in determining whether the changes are prompted by reactive oxygen species or other excited state mechanisms.

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An Anionic Porphyrin Binds β-Lactoglobulin A at a Superficial Site Rich in Lysine Residues

2009

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Binding of small ligands to globular proteins remains a major research topic in biophysics. We have studied the binding of several photoactive dyes to beta-lactoglobulin (BLG), as a model to investigate the photoinduced effects of porphyrins on proteins. A combination of optical spectroscopies (fluorescence, circular dichroism) and molecular docking simulations were used to estimate the pH-dependence of the binding parameters and the docking location for meso-tetrakis(sulfonatophenyl)-porphyrin (TPPS). We have observed that the binding of TPPS is not modulated by the pH-mediated conformational transition of the protein (i.e., Tanford transition). Binding of TPPS appears to occur with some degree of negative cooperativity. Moreover, TPPS remains bound even upon partial denaturation of the protein. These results are consistent with a superficial binding site at a location removed from the aperture of the interior beta-barrel. Binding occurs through electrostatic interactions between the negative SO(3) (-) groups of TPPS and positively charged Lys and Arg residues. This is the first study that explores the interaction of an anionic porphyrin with BLGA in a pH range that spans across the Tanford transition. Establishing the location of the binding site will enable us to explain the photoinduced conformational effects mediated by TPPS on BLG.

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Binding of Porphyrins to Tubulin Heterodimers

Cited by 25 publications

References 70 publications

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

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

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

An Anionic Porphyrin Binds β-Lactoglobulin A at a Superficial Site Rich in Lysine Residues

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