Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy.
The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct processes that occur in different GdmCl concentration ranges. From 0 to 0.5 M GdmCl, a slight alteration of the tubulin heterodimer occurs, as evidenced by a small, but reproducible increase in the rotational correlation time of the protein and a sharp decrease in the secondary structure monitored by CD. In the range 0.5-1.5 M GdmCl, significant decreases in the steady-state anisotropy and average lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl range, the number of protein molecules (labeled with Alexa 488), as determined by two-photon FCS measurements, increases by a factor of two, indicating dissociation of the tubulin dimer into monomers. From 1.5 to 4 M GdmCl, these monomers unfold, as evidenced by the continual decrease in the tryptophan steady-state anisotropy, average lifetime, and rotational correlation time, concomitant with secondary structural changes. These results help to elucidate the unfolding pathway of the tubulin heterodimer and demonstrate the value of FCS measurements in studies on oligomeric protein systems.
Sánchez
Susana A
SA
Laboratory for Fluorescence Dynamics, Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801-3080, USA.
Brunet
Juan E
JE
Jameson
David M
DM
Lagos
Rosalba
R
Monasterio
Octavio
O
D000818:Animals; D016880:Anisotropy; D001921:Brain / Q000378:metabolism; D002645:Chickens; D002942:Circular Dichroism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D019791:Guanidine / Q000737:chemistry / Q000494:pharmacology*; D006834:Hydrazines; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012399:Rotation; D013048:Specimen Handling; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014404:Tubulin / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
C417561:Alexa 488 hydrazide; D005456:Fluorescent Dyes; D006834:Hydrazines; D014404:Tubulin; D014364:Tryptophan; D019791:Guanidine