Modulation of Ultrafast Conformational Dynamics in Allosteric Interaction of Gal Repressor Protein with Different Operator DNA Sequences

Abstract

Although all forms of dynamical behaviour of a protein under allosteric interaction with effectors are predicted, little evidence of ultrafast dynamics in the interaction has been reported. Here, we demonstrate the efficacy of a combined approach involving picosecond-resolved FRET and polarisation-gated fluorescence for the exploration of ultrafast dynamics in the allosteric interaction of the Gal repressor (GalR) protein dimer with DNA operator sequences OE and OI . FRET from the single tryptophan residue to a covalently attached probe IAEDANS at a cysteine residue in the C-terminal domain of GalR shows structural perturbation and conformational dynamics during allosteric interaction. Polarisation-gated fluorescence spectroscopy of IAEDANS and another probe (FITC) covalently attached to the operator directly revealed the essential dynamics for cooperativity in the protein-protein interaction. The ultrafast resonance energy transfer from IAEDANS in the protein to FITC also revealed different dynamic flexibility in the allosteric interaction. An attempt was made to correlate the dynamic changes in the protein dimers with OE and OI with the consequent protein-protein interaction (tetramerisation) to form a DNA loop encompassing the promoter segment.

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“…The instrument response function for our TCSPC setup is measured to be ∼75 ps. Previous reports from our group depict the details of the time-resolved fluorescence measurement techniques used in our present studies. , The fluorescence anisotropy decay was obtained by using the equation, , where I VV and I VH are fluorescence transients when the emission polarizer was adjusted parallel and perpendicular to that of the excitation source. The grating factor ( G ) was determined by using a long tail matching technique.…”

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“…The time‐resolved anisotropy is defined as r (t)=( I para −G I per )/( I para +2 G I per ). The magnitude of G, the grating factor of the emission monochromator of the TCSPC system, was found using a long tail matching technique . Time‐resolved emission spectra (TRES) were constructed following the methods described earlier to determine the time‐dependent fluorescence Stokes shifts.…”

“…The magnitude of G, the grating factor of the emission monochromator of the TCSPC system, was found using al ong tail matching technique. [27] Time-resolved emission spectra (TRES) were constructed following the methods described earlier [28][29][30] to determine the time-dependent fluorescence Stokes shifts. In brief, the normalized spectral shift correlation function or the solvent correlation function, C(t), is defined as C(t) = (n t Àn 1 )/(n 0 Àn 1 ), in which n 0 , n t ,a nd n 1 are the emission peak maxima (in cm À1 )a ttime 0, t, and 1,r espectively.…”

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