title: Tubulin conformation and dynamics: a red edge excitation shift study.
abstract: The fluorescence emission maximum of a polar fluorophore in viscous medium often shows a dependence on excitation wavelength, a phenomenon which is named red edge excitation shift (REES). We have found that the fluorescence spectra of the tubulin tryptophans exhibit a REES of about 7 nm. Also, their steady state fluorescence polarization and mean lifetimes show a dependence on both excitation and emission wavelengths. These results indicate that the average tryptophan environment in tubulin is motionally restricted. Although the tryptophan(s) responsible for the observed REES effect could not be localized, it could be concluded from energy transfer experiments with the tubulin-colchicine complex that the tryptophan(s) participating in energy transfer with bound colchicine probably does not contribute to the REES. A REES of 7 nm was also observed in the case of colchicine complexed with tubulin. However, such a REES was not seen in similar studies with the B-ring analogs of colchicine, viz. 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (called AC because it lacks the B ring of colchicine) and deacetamidocolchicine (which lacks the acetamido substituent at the C-7 position of the B ring). There may be two possible reasons to explain these data. (1) Structural differences between colchicine and its analogs may give rise to differences in their excited state dipole moments which will directly affect the extent of REES, and (2) The B-ring substituent, hanging outside the colchicine binding site on the beta-subunit of the tubulin dimer, probably makes contact with the alpha-subunit of tubulin and imparts a rigidity to that region of the protein, which facilitates the REES.
authors.affiliation: Department of Biochemistry, Bose Institute, Calcutta, India.
authors.forename: S
authors.initials: S
authors.lastname: Guha
authors.forename: S S
authors.initials: SS
authors.lastname: Rawat
authors.forename: A
authors.initials: A
authors.lastname: Chattopadhyay
authors.forename: B
authors.initials: B
authors.lastname: Bhattacharyya
mesh_terms: D000818:Animals; D001665:Binding Sites; D003078:Colchicine / Q000737:chemistry / Q000378:metabolism; D004735:Energy Transfer; D005454:Fluorescence Polarization; D006041:Goats; D066298:In Vitro Techniques; D015394:Molecular Structure; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D014404:Tubulin; D014364:Tryptophan; D003078:Colchicine