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Many modern experimental techniques are based on fluorescence detection.  For studies of proteins, the application of these techniques requires labeling the protein with visible fluorescent dyes.  The objective of this project is to understand how dyes interact with proteins and how this interaction affects fluorescence measurements and specifically fluorescence polarization (FP) methods.  In FP, binding of a smaller molecule to a larger one is detected by the change in fluorescence depolarization that results from the change in volume of the rotating species.  The sensitivity of this measurement depends critically on the interaction or lack of interaction of the fluorophore with the protein, i.e. does the dye molecule “stick” to the protein or does it move freely in solution, tethered to the protein like a balloon on a string. 

As the model protein for these studies we have chosen the calcium signaling protein calmodulin (CaM), which regulates numerous enzymes by binding.  We previously reported the development of an FP assay for the binding of CaM to one of these enzymes, plasma-membrane Ca²⁺ ATPase (PMCA).  (This work has been published:  Liyanage et al., Fluorescence Polarization Assay for Calmodulin Binding to Plasma Membrane Ca²⁺–ATPase: Dependence on Enzyme and Ca²⁺ Concentrations, Anal. Biochem. 385, 1-6 (2009)). 

Surface Adsorption

For FP competition measurements involving CaM-binding peptides, we labeled the peptide M13, a CaM-binding peptide, with the dye Atto465.  We previously reported an FP measurement CaM binding to M13-Atto465.  For low sample concentrations, a serious complication in fluorescence measurements for proteins is the adsorption of the protein to the surface, leading to loss in signal.  To minimize adsorption of calmodulin in FP measurements, we tried a number of reagents to modify surface adsorption.  Figure 1 shows that the surfactant n-dodecyl-b-D-maltoside (DDM) and the protein bovine serum albumin (BSA) both impair adsorption to the surface of the peptide M13 labeled with the fluorophore Atto465.  The slight change in fluorescence intensity in the presence of DDM or BSA is comparable to the decrease in fluorescence of the free Atto465, probably due to photobleaching of the dye. 

Figure 1.  Influence of the presence of surface modifying agents on the change in fluorescence intensity of M13-Atto465.  Fluorescence intensities were measured in standard high Ca2+ HEPES buffer at pH 7.4 and 25 °C in a polystyrene cuvette. The fluorescence intensity at each time interval is an average of 10 second time trace collected at 510 nm by exciting at 453 nm.

In the presence of BSA, however, the FP of M13-Atto465 is high, suggesting interaction of M13 with BSA.  In contrast, DDM does not lead to high FP.  Thus, DDM should be useful to prevent surface adsorption.  However, we wondered whether DDM would interfere with CaM binding to M13.  Table 1 shows measurements of the dissociation constants (K_d) of CaM and M13-Atto465 measured by FP.  The results suggest a very modest affect of DDM on the K_d of CaM-M13.  

Fluorescence Polarization Assay

The competitive the dissociation constants of competing ligands can be measured without labeling each of the target ligands.  We have developed a competitive FP binding assay to measure the affinity of ligands for CaM by competition with fluorescence labeled M13.  Figure 2 shows the fluorescence anisotropy changes of M13-Atto465 upon competitive displacement by C28W (upper left panel), CKII (lower left panel), mastoparan (lower right panel), and eNOS (upper right panel).  The distinct decrease in anisotropy for the peptide C28W and the protein eNOS show competitive release of M13-Atto465.  In contrast, the CKII peptide and mastoparan do not lead to release of M13-Atto465, suggesting lower CaM affinity for these targets.  Competitive binding curves for these peptides were fit to a competitive binding model to obtain K_d. values for the competing ligands.

Figure 2.  The change in fluorescence anisotropy of M13-Atto65 bound to CaM upon competitive ligands binding to CaM.