| A model system has been developed to investigate the function of protein surface carboxylates in biomineralization. The model system consists of two components, the B1 domain of streptococcal Protein G and calcium oxalate monohydrate (COM). Site-directed mutagenesis techniques were employed to modify select aspartate and glutamate residues on the surface of Protein G. Four carboxylate variants were synthesized. Wild-type Protein G had ten carboxylates whereas Delta2 had two residues modified, D40N and D46N. Delta4 Protein G had two additional mutations, E42Q and D47Q. Delta6 Protein G had six mutations in total, D36N, D40N, E42Q, D46N, D47N, E56Q. Quantitative adsorption data was achieved by radio-detection of the labeled variants adsorbed onto COM. Extracted parameters from Langmuir data fitting showed that the number of sites available for protein binding decreased correspondingly with decreasing carboxylate number. Protein affinity for COM binding also decreased correspondingly with decreasing carboxylate number. Constant composition kinetics COM growth experiments were performed on seed crystals with preadsorbed protein. Wild-type and Delta2 Protein G promoted COM growth whereas Delta4 and Delta6 Protein G inhibited COM growth. Epi-fluorescence microscopy was utilized to determine protein adsorption location onto mature COM crystals. At low protein concentrations (∼5nM) both wild-type and Delta6 Protein G, conjugated to either fluorescein-iso-thiocyanate or tetramethyl-rhodamine-iso-thiocyanate, preferentially adsorbed to the apical faces of COM. At higher protein concentrations (∼100nM) both conjugates adsorbed to all the faces of COM. To determine the adsorbed orientation of wild-type and Delta6, electron paramagnetic resonance techniques were employed. Four cysteine mutants were synthesized, wildtype T11C, wild-type V21C, Delta6T11C, and Delta6T17C for conjugation of a methane thiosulfonate spin label via disulfide linkage. Collisional line broadening experiments with a spin relaxant indicated the approximate orientation of the adsorbed proteins. Wild-type adsorbed onto COM with the region surrounding T11C close to the COM surface and V21C distant from the COM surface. In contrast, Delta6 Protein G adsorbed onto COM with T17C close to the COM surface and T11C distant from the COM surface. |
