| Mineral-water interfacial interactions are central to biogeochemistry processes,especially microcopic dissoution and precipition(surface growth)reactions at the mineral interface,which can diretly effect mineral elemental cycling in soil geochemistry,nutrient avaliability,the fate of heavy metals,and biomineralization(the process of inorganic elements that are taken up into organisms to form minerals).The reaction rate and kinetics of each process are ultimately controlled by various organic and inorganic ions and molecules as additives(impurities)dissolved in interfacial liquids and adsorbed at the mineral interfaces.The interaction between organic macromolecules and mineral interfaces is particularly important for the crystal growth kinetics and crystal morphology modification.Using state-of-art in situ observation techinques and single-molecule force spectroscopy,it is possible to directly monitor the real-time mineral interfacial reactions at the nanoscale and the interacions between inorganic minerals-organic molecules.This will help clarify the reaction mechanisms at the mineral-water interface.In the present study,we used the calcium phosphate minerals occurring both in the soil and within organisms as mineral substrates,and chose a model protein Amelogenin's C-terminal peptide.Using in situ Atomic Force Microscopy(AFM),the dynamics of mineral-water interaction processes were observed.We also determined the binding free energy between the peptide and the Ca-P mineral surface at single-molecule level using functionalized AFM tips with single peptide molecules.In addition,the fluorescence labeling and biochemical modification studies of the peptides have finally demonstrated that the assemblies and monomers of Amelogenin's C-terminal peptides exerted different controls to regulate the kinetics and thermodynamics of interfacial growth of calcium phosphate minerals.These microscopic studies provide fundamental theroetical clues to understanding mechanisms of interfacial reactions in other complex soil minerals and biominerals.The main reaults from this study are summarized below:1.Monomeric Amelogenin's C?terminus modulates the critical length and terrace width of growth spirals on the brushite(010)face through modification of the brushite-water interfacial energies,which inhibt the sprial growth at the DCPD surface.We investigated brushite-Amelogenin's C-terminus interactions by in situ atomic force microscopy(AFM)at a series of supersaturated solutions with respect to brushite(?DCPD = 0.344-0.659).First,the growth rates(the velocities of steps along three direc-tions)of triangular sprials on DCPD(010)faces were measured in real time,and results showed that at a concentration range of 1-200 n M,Amelogenin's C-terminal peptides did not effect the velocity of moving steps,but at very low concentrations(1-10 n M)the peptide fragment significantly reduced the density(i.e.increasing the step width)of the two relatively fast steps of the [101 ]Cc and [101]Cc.This effect disappeared with increasing peptide concentrations to be more than 50 n M and the supersaturation degree ? to 0.659 from 0.344.After measuring the critical length(Lc)of the steps,we found that Lc increased with decreasing supersaturation;Lc rapidly increased with the decrease of the peptide concentrations to 1-10 n M at a constant supersaturation;no visible change in Lc was observed as peptide concentrations were greater than 50 n M.Finally,we found that at very low concentrations within a monomeric form of Amelogenin's C-terminus,the DCPD growth was modified by modulating the interface energy of the DCPD-water interfaces that affected the critical length of the steps and the step widths.This will delay the formation of steps on the growing crystal face.While at high concentrations,the peptides tend to aggregate into nanospheres which reduce the interactions between peptides and mineral surfaces.These findings provide microscopic mechanisms for controlling the formation of biominerals and soil minerals.2.The(100)surface growth of octacalcium phosphate(OCP)by nonclassical partical attachement pathway was in situ observed;During the growth processes,the elongated Amelogenin's C-terminal peptide assemblies control the morphology of the OCP nanoparticles,which led to the elongation of the spherical particles.This indicates that the assembly of Amelogenin's C-termini has the ability of modulating the surface growth of OCP crystals.We monitored the kinetics of the(100)surface growth of OCP with precisely defined thermodynamic driving forces,and found that the OCP surface growth cannot be described by classical spiral or island growth theory but in the form of particle attachement.It is different from DCPD surface growth.During in situ growth via a nonclassical particle attachment pathway,an obviously elongated aggregation of Ca-P nanoparticles induced by the assembly of Amelogenin's C-termini was observed,i.e.,the aspect ratio of the particles increased from 1:1 to 2:1 at a certain peptide concentration of 50 n M.When the peptide concentration increased to 100 n M this elongation effect disappeared due to the disassembly of the Amelogenin's C-terminal peptide on OCP crystal surface.In addition,by observing the deposition and adsorption of peptides modified by a fluorescentlabelling on the OCP crystals surface,the Amelogenin's C-terminal peptides self-assembled and disassembled on the OCP(100)faces at different peptide concentrations.The findings improve fundamental understanding of how organic molecules modulate the crystal mophology via a nonclassical crystallization route.3.The elongated self-assemble of the Amelogenin's C-terminal peptides on the OCP crystal(100)surface was clarified by AFM single-molecule force spectroscopy(SMFS).This suggests that peptides could adsorb and bind OCP nanoparticles on OCP crystal surfaces.We functionalized AFM tips with Amelogenin's C-terminal peptides,and directly quantified the binding strength of Amelogenin's C-terminal peptides on the OCP(100)surface.The peptide-decorated AFM tip was put in contact with a(100)face of an OCP crystal in phosphate buffer solutions(PBS).The force-distance curves were converted directly into the work W associtated with the breaking of the peptide-OCP bond,and finally the equilibrium free-energy,?GB =-26.0 kcal/mol,was acquired.As comparison,we also measured the force required to rupture the Amelogenin's C-terminal peptide-mica bond,and the equilibrium free-energy was only-10.3 kcal/mol.Combining in situ OCP growth kinetics with AFM SMFS futher indicated that acidic amino acids(Asp and Glu)in Amelogenin's C-terminal peptides were exposed to the surface of the peptide assembles and have the ability of binding Ca-P nanoparticles.If adsorbed onto the OCP surface of the peptide assembles,the formation of elongated calcium phosphate nanoparticles was induced.This finding sharpens our understanding of organic molecule-mineral interactions at single molecule level as well as morphology modifications during crystal growth.It provides a fundamental microscopic clue of precipitation(growth)reactions at the mineral-water interface in complex soils and other biomineralization systems. |
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