Molecular Simulations Of Protein Adsorption On Hydroxyapatite

Hydroxyapatite (Ca10(PO4)6(OH)2, HAP) has been widely used as a kind of bioactiveceramics. The physicochemical interactions between HAP surface and biomolecules arecrucial in medical applications. Many proteins help improve the osseointegration betweenHAP implants and bone tissues. Also, different morphologies of HAP nanocrystals underintricated biological templated regulation inspire the development of new biomaterials fortechnological and biomedical applications. The major contributions of this dissertation are asfollows:1. The orientation and adsorption mechanism of the10th and7-10th type III modules offibronectin (FN-III10, FN-III7-10) on HAP surfaces were investigated by a combination ofparallel tempering Monte Carlo (PTMC) and molecular dynamics (MD) methods. The PTMCresults show positively charged surface is beneficial for FN-III10and FN-III7-10adsorptionwith RGD accessible in solution, i.e., FN-III10adsorbs with “side-on” orientation andFN-III7-10adsorbs with “lying” orientation. The predictions are supported by relatedexperiments. During the adsorption studied by MD, FN-III10adsorbs on HAP surface firstdriven by Coulombic interactions at pre-adsorption stage, then by VDW interactions atpost-adsorption stage. Under weak adsorption driven by VDW interactions, FN-III10adsorbsabove the water layer. The conformational changes of adsorbed FN-III10mainly take place atits coil/loop parts. FN-III7-10experiences two stages from weak adsorption to strongadsorption when Coulombic interactions become the dominant driving force. The transition isdetermined by the anchoring of the basic residue Arg1421in the Ca2+vacancy. The moduleIII10of the flexible chain-like FN-III7-10exhibits the largest conformational change andcontributes to the adsorption most. The anchoring of the guanidine group suggests thatvacancies on biomaterials get the capacity to trap specific residues.2. The adsorption of the basic fibroblast growth factor (bFGF) on HAP (001) surface wasinvestigated by a combination of replica-exchange molecular dynamics (REMD) andconventional MD (CMD) methods. In CMD, the protein can not readily cross the surfacewater layer; while in REMD, the protein could cross the adsorption barrier from the surfacewater layer with three energetically preferred configurations, i.e., heparin-binding-up (HP-up),heparin-binding-middle (HP-middle), and heparin-binding-down (HP-down). The HP-middleorientation supports both the FGF/FGFR and DGR-integrin signaling, exhibits the strongestadsorption energy (-1149±40kJ·mol-1), owns the largest adsorption population (52.1~52.6%)and goes through the largest conformational change among the three orientations. The HP-down and HP-up orientations only support the DGR-integrin signaling, has the smalleradsorption energies of-1022±55and-894±70kJ·mol-1, own less population of27.4~27.7%and19.7~20.5%, and presents smaller conformational charge. The major population (~80%)has the protein dipole directing outwards on weakly positively charged surface. In the strongadsorption states, there are usually2~3basic residues forming the anchoring interactions of210~332kJ mol-1per residue, or accompanied by an acidic residue with adsorption energy of~207kJ mol-1. The major bound residues together form a triangle or a quadrilateral on thesurface and stabilize the adsorption geometrically, which indicate the topologic matchingsbetween the protein and HAP surfaces.3. The adsorption mechanism and adsorption free energy of a soft protein, somatomedinB (SMB) domain, on HAP (001) surface were investigated by a combination of REMD andumbrella sampling methods. The REMD method achieves enhanced sampling forenergetically favorable adsorption states and two orientations, RGD-up and RGD-middle,were obtained with52%and48%of the adsorption population respectively. The RGD-uporientation supports the cell adhesion better than the RGD-middle orientation. The SMBadsorbs on HAP surface through strong electrostatic interactions and salt bridges. TheRGD-middle orientation presents larger adsorption energy (-870±46kJ·mol-1) than theRGD-up orientation (-853±36kJ·mol-1). The SMB exhibits significant structural flexibilityin residues1-17and40-51, which is in consistent with the experimental findings. Structuralsuperpositions show that SMB exhibits lots of random coil regions in RGD-up orientation,while in RGD-middle orientation, the helix structure in residues24-28were preserved. ThePMF calculations indicate that the adsorption free energy in RGD-up orientation (-22.8±2.1kJ mol-1) is larger than it in RGD-middle orientation (-19.8±1.1kJ mol-1), which, instead ofthe adsorption energy, is in consistent with the population distribution obtained by REMD.Consequently, the RGD-up orientation is more favorable than the RGD-middle orientation,which also supports the RGD-integrin signaling better than the RGD-middle orientation.4. Different binding strengths of osteocalcin (OC) peptide to four nanostructured andsmooth HAP surfaces were investigated by MD simulations to demonstrate the underlyingmechanism of the natural biological template to direct HAP crystallization. Results show thatthe OC peptide prefers to bind energetically (-3636±223kJ·mol-1) to the concave-convexnanostructured HAP (100) surface. the strong electrostatic interactions, hydrogen bonds andionic conplementaries between OC peptide and HAP surface reveal the possible topologicalmatching on HAP (100) surface. In consistent with the experiments, OC peptide adsorbs with an open conformation to enhance the contacts with HAP surface. The underlying mechanismof biomineralization is proposed here: as the seed crystals of HAP grow in the presence of OC,the charged residues (Gla，Asp and Arg) tend to reorient, mimic and substitute the phosphateand Ca ions on (100) surface, which consequently direct the crystal to grow on the (001)surface.