Thermodynamic Roles of Basic Amino Acids in Statherin Recognition of Hydroxyapatite
Salivary statherin is a highly acidic, 43 amino acid residue protein that functions as an inhibitor of primary and secondary crystallization of the biomineral hydroxyapatite. The acidic domain at the N-terminus was previously shown to be important in the binding of statherin to hydroxyapatite surfaces. This acidic segment is followed by a basic segment whose role is unclear. In this study, the role of the basic amino acids in the hydroxyapatite adsorption thermodynamics has been determined using isothermal titration calorimetry and equilibrium adsorption isotherm analysis. Single point mutations of the basic side chains to alanine lowered the binding affinity to the surface but did not perturb the maximal surface coverage and the adsorption enthalpy. The structural and dynamic properties of the single point mutants as characterized by solid-state NMR techniques were not altered either. Simultaneous replacement of all four basic amino acids with alanine lowered the adsorption equilibrium constant by 5-fold and the maximal surface coverage by nearly 2-fold. The initial exothermic phase of adsorption exhibited by native statherin is preserved in this mutant, along with the R-helical structure and the dynamic properties of the N-terminal domain. These results help to refine the two binding site model of statherin adsorption proposed earlier in our study of wild-type statherin (Goobes, R., Goobes, G., Campbell, C.T., and Stayton, P.S. (2006) Biochemistry 45, 5576-5586). The basic charges function to reduce protein-protein charge repulsion on the HAP surface, and in their absence, there is a considerable decrease in statherin packing density on the surface at binding saturation. Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 909672
- Report Number(s):
- PNWD-SA-7813; BICHAW; TRN: US200723%%126
- Journal Information:
- Biochemistry, 46(16):4725-4733, Vol. 46, Issue 16; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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