The long-term goal of this proposal is the design and enhancement of the lubricative function of statherin with a concomitant decrease of its bacterial adhesion properties. Specifically, 15N-enriched statherin will be expressed in an optimized E. coli expression system using pGEX-2T as the cloning vehicle. The solution structure of the purified 15N-enriched statherin will be determined using three-dimensional nuclear magnetic resonance spectroscopy. Fluorescence and FTIR spectroscopy will be used to test whether statherin undergoes any conformational change upon binding to hydroxyapatite. The information gained from these biophysical studies will be used with molecular modeling to deduce the three-dimensional structure of statherin at the enamel interface. Using the model at the interface, specific residues and secondary structural motifs will be selected for substitution using site-directed mutagenesis. The hypothesis is that statherin requires a highly polar N-terminal region to anchor itself at the interface and the C-terminal segment for both lubricity, as well as bacterial attachment. The lubrication property of statherin is dependent upon its ability to provide an amphipathic film upon binding to the enamel surface. This hypothesis will be tested by specifically preparing several analogs of statherin in which the hydrophobicity of the C-terminal region will be increased by individually replacing the tyrosine residues with more hydrophobic residues such as Phe, Val, Ile and Leu. All these analogs will be produced by site-directed mutagenesis. The efficacy of the analogs to enhance lubrication and their ability to decrease the attachment of Actinomyces viscosus to analog-coated hydroxyapatite will be examined. Information from the behavior of these analogs will allow the design and production of an analog with optimum lubrication but minimal bacterial adhesion properties.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Specialized Center (P50)
Project #
5P50DE008240-10
Application #
6296257
Study Section
Project Start
1996-09-01
Project End
1999-11-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
10
Fiscal Year
1996
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Type
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
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