Single–Molecule Determinations Of Protein–Mineral Binding Energies And Kinetics Of Surface Growth And Phase Transformation Of Calcium Phosphates Modulated By Amelotin Proteins

Biomineralization is a process of forming inorganic minerals under the control of organic molecules inside or outside organisms.The research area of biomineralization is an interdisciplinary field that includes agricultural,environmental,medical,dental,and materials science.Fundamentally,biomineralization is mainly the interaction and reaction between organic molecules and inorganic minerals.Hence,it is particularly important to research the mechanism of mineral formation and organic molecule interactions in biomineralization.Organic molecules play an important role in biomineralization,and biomolecules are diverse in structure,composition,morphology and function,depending on organic molecules.However,the investigation of interactions of organic molecules and nucleus or specific crystal surface during the earliest nucleation stage and following crystal growth kinetics is still insufficient,which is mainly due to the lack of in situ research tools.In our study,we use in situ monitoring technologies to observe the dynamic growth and phase transition process of inorganic minerals at the nanoscale,as well as the interaction of organic molecules and inorganic minerals in the reaction process,which can help us to understand the formation mechanism of biominerals.Calcium phosphate(Ca-P)minerals,as one of the most common biominerals,mainly form biological hard tissues as biological scaffolds or protectors(such as bone and teeth of animals and trichomes of plants).Meanwhile,the mineralization of calcium phosphate also plays an important role in the transport of calcium,phosphorus and heavy metal in environment.In the crystallization process of Ca-P,Amelotin(AMTN)is a newly discovered protein which can promote the crystallization of calcium phosphate.Therefore,we synthesized different calcium phosphate phases under laboratory conditions,and recombinant Amelotin using Escherichia coli to provide effective materials for our research.We used in situ atomic force microscopy(AFM)to directly image the surface growth of brushite(DCPD)in the presence of recombinant AMTN.By the use of high-resolution Raman spectroscopy and AFM,we directly observe time-resolved in situ amorphous calcium phosphate(ACP)phase transformation,and the effect AMTN in this process.In addition,we used single-molecule force spectroscopy(SMFS)to explore the thermodynamic mechanism of the interaction of AMTN and DCPD/ACP.The main conclusions are as follows:1.Synthesis and purification of recombinant Amelotin(AMTN).Escherichia coli were used to express recombinant AMTN protein.Expression levels of recombinant AMTN can be improved in E.coli by using an N-terminal histidine tag.By histidine tag,we can easily and quickly obtain high purity proteins.Moreover,AMTN mutants were synthesized by direct deletion of a gene sequence from the plasmid by PCR.This method can be used not only to synthesize AMTN,but also to synthesize other proteins or peptides,providing protein biomaterials for studying the interaction between organic molecules and calcium phosphate minerals.2.Amelotin(AMTN)regulates the nanoscale growth kinetics of brushite(DCPD)through completely different action mechanisms at varied protein concentrations,and the active SSEEL subdomain in AMTN may contribute to a specific interaction with the DCPD(010)face.We used in situ atomic force microscopy(AFM)to directly image the surface growth of brushite(dicalcium phosphate dihydrate,DCPD,Ca HPO4·2H2O)in the presence of recombinant human AMTN.Measured step movement velocities of the DCPD(010)face show that AMTN promotes crystal face growth only within a limited concentration range,whereas inhibition occurs outside of this range.A peptide derived from a highly conserved and potentially phosphorylated motif SSEEL(Ser-Ser-Glu-Glu-Leu)within the AMTN inhibits crystal growth similar to that of the AMTN at low concentration.By the use of single-molecule force spectroscopy(SMFS),we directly measure the binding of the full-length AMTN and SSEEL to the DCPD(010)face.The similar rupture forces reveal that this active SSEEL subdomain may contribute to a specific interaction with the DCPD(010)face,despite significant differences in binding energies of the full-length AMTN and SSEEL peptides to the DCPD surfaces.The findings reveal the kinetic and energetic basis for modulation of the Ca-P crystal face growth by AMTN.3.AMTN facilitates the formation of HAP crystals by promoting phase transformation from amorphous calcium phosphate(ACP).Single-molecule measurements identify a highly conserved SSEEL motif that controls phase transition of calcium phosphates.With the use of high-resolution Raman spectroscopy and atomic force microscopy to directly observe in situ ACP crystallization,we show that AMTN facilitates the formation of HAP crystals by promoting phase transformation from ACP.In the presence of a near-physiological fluid,mineral conversions of ACP proceed through dissolution and recrystallization at the ACP-fluid interface,where the adsorbed AMTN promotes the ACP dissolution through a strong complexation of the AMTN with calcium ions, occurring even in supersaturated solutions.Single-molecule measurements of equilibrium rupture force and the binding free-energy of AMTN adsorbed to four calcium phosphate phases(ACP,DCPD,OCP and HAP)identify a highly conserved SSEEL motif that controls phase transition of calcium phosphates with enhanced mechanical properties.4.The organized assembly of fluoro-substituted hydroxyapatite is controlled by both Amelotin and F-ions during the phase transition from an amorphous calcium phosphate precursor phase to the final crystallization product with enhanced mechanical properties.We used in situ atomic force microscopy(AFM)to image the transition from an amorphous calcium phosphate(ACP)precursor phase to fluoro-substituted HAP(F-HAP)under the influence of AMTN.In the presence of F-ions only,F-HAP nanorods formed randomly on the ACP surface,but assembled in an orderly manner in the presence of 1 ?M AMTN.Using transmission electron microscopy(TEM),we observed a stepwise nucleation mechanism from pre-nucleation clusters to rod-like crystals templated and modulated by AMTN.Mechanical measurements of nanorods surface revealed the enhanced modulus of the layers with organized nanorods of F-HAP.Our findings emphasize that AMTN plays an important role in modulating crystallization and organization of F-HAP crystals,which is significant for potential synthesis of Ca-P materials and the study of detaining of toxic fluoride ions in the environment.