Exploring the hydrophobic binding sites of calmodulin with fluorescent probes: Dynamic formation of the excimer

1170-1183Molecular probes consisting of hydrophobic and hydrophilic functional groups separated by a flexible linker have been investigated for their binding with calmodulin, a ubiquitous calcium binding protein. For example, (9anthryl)methylamine HCl (AMAC), 3-(9-anthry l)propylamine HCl (APAC), N-ethyl-(9-anthryl)methylamine HCl, (N-Et-AMAC),and 4-( I-pyrenyl)butylamine HCl (PBAC) bind to calmodulin. The cationic and hydrophobic moieties of these probes separated by a linker provide multiple sites for interaction with the protein. Length and flexibility of the linker play a key role in the binding. For example, the isomeric probes N-Et-AMAC and APAC show major differences in their binding interactions. Probes with long linkers (APAC and PBAC) bind better than the probes with short linkers (AMAC and N-Et-AMAC). The electronic absorption spectra of the anthryl probes AMAC, APAC and N-Et-AMAC undergo only minor changes upon binding to calmodium . In contrast, the absorption spectrum of the pyrenyl analog, PBAC,undergoes a large red shift (~ 6 nm) and ~20% decrease in the extinction coefficients. Such a red shift is consistent with the binding of the pyrenyl moiety to hydrophobic regions of calmodulin. The decrease in the absorption is indicative of stacking interactions between the pyrenyl chromophore and the aromatic residues of the protein. The fluorescence spectra are red shifted, accompanied by quenching of the overall intensity in the cases of AMAC, N-Et-AMAC or APAC. Binding of PBAC to calmodulin results in a new emission band at 475 nm assigned to the pyrene excimer, consistent with the literature reports. Fluorescence quenching experiments with potassium iodide coupled with lifetime measurements indicate. that the calmodulin-bound probes are significantly protected from the aqueous phase. The decreases in the quenching constants are 42, 22 and 69% for APAC, N-Et-AMAC and PBAC when compared to those of the corresponding free probes, respectively. The extent of protection from iodide depends upon the length of the linker and hydrophobicity. The observed order of protection, PBA>> APAC>AMAC>N-Et-AMAC, parallels the hydrophobicity of the aromatic moiety as well as the length of the linker. Binding of PBAC to calmodulin results in induced circular dichroism bands, establishing the asymmetric environment of the bound probe. Time-resolved fluorescence studies indicate that the protein-bound probes exhibit multi-component decays with lifetimes, 7.7 and 11.6 ns for APAC; 2.0 and 11ns for N-Et-AMAC; 13 , 80 and 198 ns for PBAC. Time-resolved emission spectra of PBAC bound to calmodulin clearly show the growth and decay of the excimer emission with a maximum around 475 nm. The excimer grows with a time constant of 12 ns and the growth rate is independent of the protein concentration. No such excimer emission is observed with the anthryl probes. The excimer formation is found to depend upon temperature, with an activation barrier of 53 kJ/mol. The magnitude of the barrier and the excimer growth rate constant suggest that the excimer formation is perhaps controlled by the local segmental motion or bending of the protein. If so, the present suggest a dynamic flexible structure for calmodulin, in aqueous solutions. In general, the binding is enhanced with the increase in hydrophobicity of the probe as well as length of the linker separating the hydrophobic and hydrophilic moieties.