Conformation of microtubule-bound paclitaxel determined by fluorescence spectroscopy and REDOR NMR

Yankun Li, Barbara Poliks, Lynette Cegelski, Mark Poliks, Zygmunt Gryczynski, Grzegorz Piszczek, Prakash G. Jagtap, Daniel R. Studelska, David G.I. Kingston, Jacob Schaefer, Susan Bane

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Abstract

The conformation of microtubule-bound paclitaxel has been examined by fluorescence and solid-state NMR spectroscopy. A fluorescent derivative of paclitaxel, 3'-N-debenzoyl-3'-N-(m-aminobenzoyl)paclitaxel (N-AB-PT), was prepared by semisynthesis. No differences in the microtubule-promoting activity between N-AB-PT and paclitaxel were observed, demonstrating that addition of the amino group did not adversely affect the ligand-receptor association. The distance between the fluorophore N-AB-PT and the colchicine binding site on tubulin polymers was determined through time-resolved measurements of fluorescence resonance energy transfer to be 29 ± 2 Å. The absorption and emission spectra of N-AB-PT bound to microtubules and in various solvents were measured. A plot of the stokes shift as a function of solvent polarity was highly unusual. The Stokes shift increased linearly with solvent polarity in protic solvents, which is expected due to the nature of the fluorophore. In aprotic solvents, however, the Stokes shift was invariant with solvent polarity, indicating that the fluorophore was somehow shielded from the effects of the solvent. These data are best explained by considering the solution-state conformational properties of paclitaxel. It is known that paclitaxel adopts different conformations depending on the nature of the solvent, and these fluorescence data are consistent with the molecule adopting a 'hydrophobic collapsed' conformation in protic solvents and an 'extended' conformation in aprotic solvents. The Stokes shift of microtubule- bound N-AB-PT was within the protic solvent region, demonstrating that microtubule-bound paclitaxel is in a hydrophobic collapsed conformation. Microtubule-bound paclitaxel was also investigated by solid-state NMR. Paclitaxel was labeled with 19F at the para position of the C-2 benzoyl substituent and with 13C and 15N in the side chain. Distances between the fluorine and carbon nuclei were determined by REDOR. The distance between the fluorine and the 3'-amide carbonyl carbon was 9.8 ± 0.5 Å, and the distance between the fluorine atom and the 3'-methine carbon was 10.3 ± 0.5 Å. These spectroscopic data were used in conjunction with molecular modeling to refine the microtubule-bound conformation of paclitaxel and to suggest an alternative orientation of the ligand within the paclitaxel binding site.

Original languageEnglish
Pages (from-to)281-291
Number of pages11
JournalBiochemistry
Volume39
Issue number2
DOIs
StatePublished - 18 Jan 2000

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Fluorescence Spectrometry
Fluorescence spectroscopy
Paclitaxel
Microtubules
Conformations
Nuclear magnetic resonance
Fluorine
Fluorophores
Carbon
Fluorescence
Binding Sites
Ligands
Fluorescence Resonance Energy Transfer
Molecular modeling
Colchicine
Tubulin
Time measurement
Amides
Crystal orientation
Nuclear magnetic resonance spectroscopy

Cite this

Li, Yankun ; Poliks, Barbara ; Cegelski, Lynette ; Poliks, Mark ; Gryczynski, Zygmunt ; Piszczek, Grzegorz ; Jagtap, Prakash G. ; Studelska, Daniel R. ; Kingston, David G.I. ; Schaefer, Jacob ; Bane, Susan. / Conformation of microtubule-bound paclitaxel determined by fluorescence spectroscopy and REDOR NMR. In: Biochemistry. 2000 ; Vol. 39, No. 2. pp. 281-291.
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abstract = "The conformation of microtubule-bound paclitaxel has been examined by fluorescence and solid-state NMR spectroscopy. A fluorescent derivative of paclitaxel, 3'-N-debenzoyl-3'-N-(m-aminobenzoyl)paclitaxel (N-AB-PT), was prepared by semisynthesis. No differences in the microtubule-promoting activity between N-AB-PT and paclitaxel were observed, demonstrating that addition of the amino group did not adversely affect the ligand-receptor association. The distance between the fluorophore N-AB-PT and the colchicine binding site on tubulin polymers was determined through time-resolved measurements of fluorescence resonance energy transfer to be 29 ± 2 {\AA}. The absorption and emission spectra of N-AB-PT bound to microtubules and in various solvents were measured. A plot of the stokes shift as a function of solvent polarity was highly unusual. The Stokes shift increased linearly with solvent polarity in protic solvents, which is expected due to the nature of the fluorophore. In aprotic solvents, however, the Stokes shift was invariant with solvent polarity, indicating that the fluorophore was somehow shielded from the effects of the solvent. These data are best explained by considering the solution-state conformational properties of paclitaxel. It is known that paclitaxel adopts different conformations depending on the nature of the solvent, and these fluorescence data are consistent with the molecule adopting a 'hydrophobic collapsed' conformation in protic solvents and an 'extended' conformation in aprotic solvents. The Stokes shift of microtubule- bound N-AB-PT was within the protic solvent region, demonstrating that microtubule-bound paclitaxel is in a hydrophobic collapsed conformation. Microtubule-bound paclitaxel was also investigated by solid-state NMR. Paclitaxel was labeled with 19F at the para position of the C-2 benzoyl substituent and with 13C and 15N in the side chain. Distances between the fluorine and carbon nuclei were determined by REDOR. The distance between the fluorine and the 3'-amide carbonyl carbon was 9.8 ± 0.5 {\AA}, and the distance between the fluorine atom and the 3'-methine carbon was 10.3 ± 0.5 {\AA}. These spectroscopic data were used in conjunction with molecular modeling to refine the microtubule-bound conformation of paclitaxel and to suggest an alternative orientation of the ligand within the paclitaxel binding site.",
author = "Yankun Li and Barbara Poliks and Lynette Cegelski and Mark Poliks and Zygmunt Gryczynski and Grzegorz Piszczek and Jagtap, {Prakash G.} and Studelska, {Daniel R.} and Kingston, {David G.I.} and Jacob Schaefer and Susan Bane",
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Li, Y, Poliks, B, Cegelski, L, Poliks, M, Gryczynski, Z, Piszczek, G, Jagtap, PG, Studelska, DR, Kingston, DGI, Schaefer, J & Bane, S 2000, 'Conformation of microtubule-bound paclitaxel determined by fluorescence spectroscopy and REDOR NMR', Biochemistry, vol. 39, no. 2, pp. 281-291. https://doi.org/10.1021/bi991936r

Conformation of microtubule-bound paclitaxel determined by fluorescence spectroscopy and REDOR NMR. / Li, Yankun; Poliks, Barbara; Cegelski, Lynette; Poliks, Mark; Gryczynski, Zygmunt; Piszczek, Grzegorz; Jagtap, Prakash G.; Studelska, Daniel R.; Kingston, David G.I.; Schaefer, Jacob; Bane, Susan.

In: Biochemistry, Vol. 39, No. 2, 18.01.2000, p. 281-291.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Conformation of microtubule-bound paclitaxel determined by fluorescence spectroscopy and REDOR NMR

AU - Li, Yankun

AU - Poliks, Barbara

AU - Cegelski, Lynette

AU - Poliks, Mark

AU - Gryczynski, Zygmunt

AU - Piszczek, Grzegorz

AU - Jagtap, Prakash G.

AU - Studelska, Daniel R.

AU - Kingston, David G.I.

AU - Schaefer, Jacob

AU - Bane, Susan

PY - 2000/1/18

Y1 - 2000/1/18

N2 - The conformation of microtubule-bound paclitaxel has been examined by fluorescence and solid-state NMR spectroscopy. A fluorescent derivative of paclitaxel, 3'-N-debenzoyl-3'-N-(m-aminobenzoyl)paclitaxel (N-AB-PT), was prepared by semisynthesis. No differences in the microtubule-promoting activity between N-AB-PT and paclitaxel were observed, demonstrating that addition of the amino group did not adversely affect the ligand-receptor association. The distance between the fluorophore N-AB-PT and the colchicine binding site on tubulin polymers was determined through time-resolved measurements of fluorescence resonance energy transfer to be 29 ± 2 Å. The absorption and emission spectra of N-AB-PT bound to microtubules and in various solvents were measured. A plot of the stokes shift as a function of solvent polarity was highly unusual. The Stokes shift increased linearly with solvent polarity in protic solvents, which is expected due to the nature of the fluorophore. In aprotic solvents, however, the Stokes shift was invariant with solvent polarity, indicating that the fluorophore was somehow shielded from the effects of the solvent. These data are best explained by considering the solution-state conformational properties of paclitaxel. It is known that paclitaxel adopts different conformations depending on the nature of the solvent, and these fluorescence data are consistent with the molecule adopting a 'hydrophobic collapsed' conformation in protic solvents and an 'extended' conformation in aprotic solvents. The Stokes shift of microtubule- bound N-AB-PT was within the protic solvent region, demonstrating that microtubule-bound paclitaxel is in a hydrophobic collapsed conformation. Microtubule-bound paclitaxel was also investigated by solid-state NMR. Paclitaxel was labeled with 19F at the para position of the C-2 benzoyl substituent and with 13C and 15N in the side chain. Distances between the fluorine and carbon nuclei were determined by REDOR. The distance between the fluorine and the 3'-amide carbonyl carbon was 9.8 ± 0.5 Å, and the distance between the fluorine atom and the 3'-methine carbon was 10.3 ± 0.5 Å. These spectroscopic data were used in conjunction with molecular modeling to refine the microtubule-bound conformation of paclitaxel and to suggest an alternative orientation of the ligand within the paclitaxel binding site.

AB - The conformation of microtubule-bound paclitaxel has been examined by fluorescence and solid-state NMR spectroscopy. A fluorescent derivative of paclitaxel, 3'-N-debenzoyl-3'-N-(m-aminobenzoyl)paclitaxel (N-AB-PT), was prepared by semisynthesis. No differences in the microtubule-promoting activity between N-AB-PT and paclitaxel were observed, demonstrating that addition of the amino group did not adversely affect the ligand-receptor association. The distance between the fluorophore N-AB-PT and the colchicine binding site on tubulin polymers was determined through time-resolved measurements of fluorescence resonance energy transfer to be 29 ± 2 Å. The absorption and emission spectra of N-AB-PT bound to microtubules and in various solvents were measured. A plot of the stokes shift as a function of solvent polarity was highly unusual. The Stokes shift increased linearly with solvent polarity in protic solvents, which is expected due to the nature of the fluorophore. In aprotic solvents, however, the Stokes shift was invariant with solvent polarity, indicating that the fluorophore was somehow shielded from the effects of the solvent. These data are best explained by considering the solution-state conformational properties of paclitaxel. It is known that paclitaxel adopts different conformations depending on the nature of the solvent, and these fluorescence data are consistent with the molecule adopting a 'hydrophobic collapsed' conformation in protic solvents and an 'extended' conformation in aprotic solvents. The Stokes shift of microtubule- bound N-AB-PT was within the protic solvent region, demonstrating that microtubule-bound paclitaxel is in a hydrophobic collapsed conformation. Microtubule-bound paclitaxel was also investigated by solid-state NMR. Paclitaxel was labeled with 19F at the para position of the C-2 benzoyl substituent and with 13C and 15N in the side chain. Distances between the fluorine and carbon nuclei were determined by REDOR. The distance between the fluorine and the 3'-amide carbonyl carbon was 9.8 ± 0.5 Å, and the distance between the fluorine atom and the 3'-methine carbon was 10.3 ± 0.5 Å. These spectroscopic data were used in conjunction with molecular modeling to refine the microtubule-bound conformation of paclitaxel and to suggest an alternative orientation of the ligand within the paclitaxel binding site.

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DO - 10.1021/bi991936r

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