AVS 57th International Symposium & Exhibition
    Nanometer-scale Science and Technology Thursday Sessions
       Session NS+BI-ThA

Paper NS+BI-ThA8
Probing Biomineralization Protein Interactions with Hydroxyapatite Using SFG and NEXAFS Spectroscopy

Thursday, October 21, 2010, 4:20 pm, Room La Cienega

Session: Biomolecular Templates & Bioinspired Nanomaterials
Presenter: T.M. Weidner, University of Washington
Authors: T.M. Weidner, University of Washington
M. Dubey, University of Washington
N.F. Breen, University of Washington
J. Ash, University of Washington
J.E. Baio, University of Washington
C. Jaye, National Institute of Standards and Technology
D.A. Fischer, National Institute of Standards and Technology
G.P. Drobny, University of Washington
D.G. Castner, University of Washington
Correspondent: Click to Email
The structural integrity of hydroxyapatite (HAP) in tooth enamel is maintained through the saliva environment that is supersaturated with calcium and phosphate salts. The biomineralization protein statherin adsorbs onto HAP surfaces with high binding affinity. It regulates HAP growth and prevents the buildup of excess HAP on the tooth surface by inhibiting spontaneous calcium phosphate growth. Owing to the importance of the underlying physiological processes and a general interest in biomineralization mechanisms, the binding of statherin to HAP has attracted significant interest in the biomaterials community. Sum frequency generation (SFG) spectroscopy can probe protein orientation and secondary structure at the solid-liquid interface and we have recently shown it can address specific protein regions with atomic resolution when combined with isotopic labeling.1 Near edge X-ray absorption fine structure (NEXAFS) spectroscopy can give valuable information about the structure and binding chemistry of proteins on surface. We have combined both techniques to characterize the structure of the binding domain of statherin, SN15, a short peptide with 15 residues (Ac-DSSEENKFLRRIGRFG-OH) adsorbed onto a model HAP surface. Protein adsorption was verified using X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. SFG confirmed a loosely helical secondary structure of SN15 on HAP. Deuteration was used to specifically probe the orientations of the hydrophobic leucine and isoleucine side chains with SFG in situ. Side-chain orientations were determined using ratios of the symmetric and asymmetric CD3 stretching modes. The leucine chain was tilted 120° from the surface normal (pointing towards the surface) and the isoleucine was tilted 5° from the surface normal. For the first time, element labels were employed to probe individual side chain orientations with NEXAFS spectroscopy. Para- and perfluorination of the phenylalanine rings F7 and F14 allowed us to precisely measure their orientations using angle dependent NEXAFS data. The tilt angles from the surface normal were determined to be 26° for F7 and 35° for F14.

[1] T. Weidner, N. F. Breen, K. Li, G. P. Drobny, D G. Castner, submitted.