Quantification of the binding affinity of a specific hydroxyapatite binding peptide.
The genesis of bone and teeth involves highly coordinated processes, which involve multiple cell types and proteins that direct the nucleation and crystallization of inorganic hydroxyapatite (HA). Recent studies have shown that peptides mediate the nucleation process, control HA microstructure or even inhibit HA mineralization. Using phage display technology, a short peptide was identified that binds to crystalline HA and to HA-containing domains of human teeth with chemical and morphological specificity. However, the binding affinity and specific amino acids that significantly contribute to this interaction require further investigation. In this study, we employ a microfluidic chip based surface plasmon resonance imaging (SPRi) technique to quantitatively measure peptide affinity by fabricating a novel 4 layer HA SPR sensor. We find the peptide (SVSVGMKPSPRPGGGK) binds with relatively high affinity (K(D) = 14.1 microM +/- 3.8 microM) to HA. The independently measured amino acid fragment SVSV seems to impart a significant contribution to this interaction while the MKPSP fragment may provide a conformational dependent component that enhances the peptides affinity but by itself shows little specificity in the current context. These data show that together, the two moieties promote a stronger synergistic binding interaction to HA than the simple combination of the individual components.
Weiger
Michael C
MC
Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Park
Jung Jin
JJ
Roy
Marc D
MD
Stafford
Christopher M
CM
Karim
Alamgir
A
Becker
Matthew L
ML
D000595:Amino Acid Sequence; D003460:Crystallization; D017886:Durapatite / Q000378:metabolism*; D006801:Humans; D046210:Microfluidic Analytical Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods; D014070:Tooth / Q000737:chemistry
D010446:Peptide Fragments; D019151:Peptide Library; D010455:Peptides; D017886:Durapatite