Investigation of Dye-Protein Interactions and Optimization of Fluorescence-Based Assays for Target Binding of Calmodulin

44160-AC4
Investigation of Dye-Protein Interactions and Optimization of Fluorescence-Based Assays for Target Binding of Calmodulin

Carey K. Johnson, University of Kansas

Chemists and biologists frequently turn to fluorescence techniques to probe protein interactions and dynamics. These techniques often rely on a fluorescent dye covalently attached to the protein. The nature of interactions between the dye and the protein are important for the reliability of these assays. The goals of this project are (1) to characterize the influence of electrostatic interactions on the interactions of dyes attached to the protein calmodulin (CaM), and (2) to use this knowledge to develop sensitive assays for target binding by CaM. Dye-protein interactions in CaM. The fluorescence lifetime and fluorescence anisotropy decays were measured for CaM labeled with three fluorescence dyes bearing different charges by time-correlated single photon counting. The following dyes were chosen in this experiment.

Dye	Charge	Wavelength abs/emission (nm)	rotational correlation time (ns)	Fraction
Alexa Fluor488	-1	453/460	0.19	0.51
			3.59	0.49
Oregon Green 488	0	475/460	0.20	0.50
			4.18	0.50
Atto465	1	475/460	0.73	0.29
			7.95	0.71

The results of the time resolved anisotropy measurements indicate that there is a direct involvement of the charge of the dye in determining dye-protein interactions. The positively charged Atto465 has a longer rotational time with the larger fraction indicating that these dye molecules tend to stick to the protein. On the other hand negatively charged AF488 and uncharged Oregon Green 488 have lower fractions for the longer rotational times indicating low sticking of the dye to protein.

Assays of CaM-target binding affinity. Based on the above results, we used CaM labeled with Atto465 for a fluorescence polarization (FP) assay of CaM binding to plasma-membrane Ca²⁺-ATPase (“PMCA,” a Ca²⁺ pump). The larger anisotropy of Atto465 compared to the other dyes investigated results in increased sensitivity in FP measurements. Changes in the steady-state anisotropy and were fit by nonlinear regression to determine the dissociation

constant K_d for the CaM- PMCA complex. The result shown in the figure yields the binding affinity of CaM-PMCA (K_d = 6.9 nM). As the PMCA concentration was increased above 12 nM, a further increase in the anisotropy indicated aggregation of the protein. The second figure shows the results of FP measurements as a function of Ca²⁺ concentration have similarly yielded the Ca²⁺ affinity of CaM-PMCA complexes. The dependence at Ca²⁺ concentrations below 200 nM shows cooperativity in Ca²⁺ binding (Hill coefficient 1.6). A manuscript has been submitted describing these results.

44160-AC4 Investigation of Dye-Protein Interactions and Optimization of Fluorescence-Based Assays for Target Binding of Calmodulin

44160-AC4
Investigation of Dye-Protein Interactions and Optimization of Fluorescence-Based Assays for Target Binding of Calmodulin