Analysis of Protein-Mineral Interactions by Fluorescence Recovery after Photobleaching

Objectives: Biological mineral growth, habit (crystal size and shape) and orientation, is controlled in part by the adsorption of extracellular macromolecules to specific crystal faces and edges. Phosphopeptides of osteopontin (OPN) have been shown to adsorb to {100} faces of calcium oxalate monohydrate (COM) and to inhibit growth of the crystal in 〈100〉 direction. The inhibitory potency is positively correlated to the phosphate content of the peptide. Molecular dynamics (MD) studies suggest that phosphate groups stabilize the peptide-mineral interaction. Experimental quantification of the strength of these interactions would validate the data derived from MD.

Methods: Fluorescence recovery after photobleaching (FRAP) has been widely used to investigate diffusion reactions in biological membranes. Recently, a systematic approach has been developed to extract kinetic information from FRAP experiments. We apply this approach to study adsorption/desorption reactions of synthetic peptides corresponding to amino acids 220-235 of rat bone OPN containing 0, 1, or 3 phosphate groups (P0, P1, P3) with the {100} faces of COM. Additionally, we calculated free energy profiles of the adsorption/desorption process using an adaptive biasing force (ABF) MD approach.

Results: Analysis of the results show a decrease of the desorption rate associated with increased phosphorylation. K_off values for P0, P1, and P3 were found to be 0.12 sec^-1, 0.02 sec^-1, and 0.002 sec^-1, respectively. Changes in free energy between the adsorbed and desorbed states were calculated to be 250 kJ/mol, 326 kJ/mol, and 355 kJ/mol respectively. These values correspond well to the obtained rate constants.

Conclusions: The results from this study show that phosphorylation enhances a stable mineral interaction. This study also demonstrates the effective use of FRAP analysis and ABF MD to study the kinetics of protein-mineral interactions.

Supported by CIHR and NSERC.