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.

The feasibility of coherent energy transfer in microtubules

“…Calculated site energies using a dielectric constant value of 8.41 range between 35 888 and 36 276 cm 21 , giving the maximum energy difference between Trps of 388 cm 21 . These site energies are shown in figure 1c and is there a significant delocalization over more than one pigment, with meaningful contributions between Trps 4 and 7, and Trps 2 and 3, owing to the close site energies between these pigments and their relatively large couplings.…”

“…These are comparable to the room temperature fluorescence quantum yields of bacteriochlorophyll of 0.18, and the yield for LH1 of 0.08 and LH2 of 0.10 [20], respectively. The 'red edge effect' has also been observed in tubulin [21] indicating Trp to Trp energy transfer [22]. Thus, these may serve as potential conduction pathways in tubulin and microtubules [23].…”

Micellar Organization and Dynamics: A Wavelength-Selective Fluorescence Approach

“…Wavelength-selective fluorescence has been previously applied for monitoring protein dynamics and conformation in solution 19,[67][68][69][70][71] as well as in more complex systems such as the intact eye lens. 72, 73 We have previously applied this approach for studying organization and dynamics of membrane-bound probes and peptides.…”

Novel Insights Into Protein Structure and Dynamics Utilizing the Red Edge Excitation Shift Approach

“…The tryptophan residue in these cases is exposed to aqueous environment undergoing much faster solvent relaxation as compared to the fluorescence lifetimes of the concerned tryptophans. It is for this reason that tryptophans in denatured proteins do not exhibit REES, e.g., tubulin 77 (see later). However, considerable REES effects were observed for many proteins (such as tetrameric form of melittin, human serum albumin, albumin complexed with sodium dodecyl sulfate) with intermediate maxima of emission (between 325 and 341 nm).…”

“…80 The REES approach has been used in a number of cases to monitor the tryptophan environment and dynamics in proteins such as human α 1 -acid glycoprotein, 81 bothropstoxin-I from the venom of Bothrops jararacussu, 82 ascorbate oxidase, 83 smooth muscle myosin light chain kinase, 84 skeletal myosin rod, 85 the human leukocyte antigen complex, 86 and the pore-forming α-toxin from Staphylococcus aureus. 87 In addition, it has been shown that the environment of tryptophans in cytoskeletal proteins such as tubulin 77 and spectrin 88,89 are motionally restricted and display REES. Importantly, observation of REES in multitryptophan proteins considerably rules out the possibility of homotransfer among tryptophans.…”