Abstract
Purpose: :
To further develop the mechanistic understanding of α–crystallin chaperone activity, we investigate the nature of the substrate intermediate states recognized and the conformations that are stably bound. For this purpose, we introduced bimane/tryptophan pairs in selected regions of T4 Lysozyme and determined their proximity and the emission characteristics of the bimane in the bound state.
Methods: :
The mutants were introduced into the L99A background, a mutation in the hydrophobic core that reduces the free energy of unfolding and promotes association to α–crystallin. The bimane/Trp double mutants were selected to fingerprint the tertiary fold of the N and C terminal domains and their packing interface. The distances between the two sites are within the range previously shown to result in distance–dependent quenching. The corresponding single cysteine mutants were also constructed. Fluorescence spectroscopy was used to monitor the binding of the various T4L–L99A mutants to αB–Crystallin. Binding isotherms were obtained by incubating defined ratios of αB–Crystallin:T4L–L99A mutants and collecting the emission spectrum of the bimane label.
Results: :
For all double mutants, bimane fluorescence emission is quenched by the presence of Trp, consistent with close proximity in the unbound, folded state of T4L. Upon binding to αB–Crystallin, bimane fluorescence emission intensity recovers to the same level of emission as for the bimane alone, indicating an increase in distance between bimane and Trp. The increase in distance was reported by pairs distributed across the molecule consistent with global changes in structure. A characteristic blue shift in max of the bimane emission spectrum also accompanies binding, indicating movement of bimane into a more hydrophobic environment. The magnitude of the shift is dependent on the location of the site where the bimane label is attached. Overall, sites in the C–terminus in the neighborhood of L99A showed a larger blue shift than sites in the N–terminal domain.
Conclusions: :
The increase in bimane fluorescence upon binding indicates that the bimane/Trp separation has increased beyond 15 angstroms. In conjunction with previous studies, this indicates that stably bound T4L is extensively unfolded. The pattern of decreased quenching is independent of the mode of binding suggesting similar conformation of bound T4L in the two modes. The polarized distribution of the blue shift in max is consistent with preferential binding of the T4L molecule with the N–terminal domain more accessible to the solvent.
Keywords: chaperones • crystalline lens • cataract