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Title: Thermodynamic Roles of Basic Amino Acids in Statherin Recognition of Hydroxyapatite

Abstract

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 adsorptionmore » 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.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
909672
Report Number(s):
PNWD-SA-7813
Journal ID: ISSN 0006-2960; BICHAW; TRN: US200723%%126
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry, 46(16):4725-4733; Journal Volume: 46; Journal Issue: 16
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ADSORPTION; ADSORPTION ISOTHERMS; AMINO ACIDS; GENE MUTATIONS; THERMODYNAMICS

Citation Formats

Goobes, Rivka, Goobes, Gil, Shaw, Wendy J., Drobny, Gary P., Campbell, Charles T., and Stayton, Patrick. Thermodynamic Roles of Basic Amino Acids in Statherin Recognition of Hydroxyapatite. United States: N. p., 2007. Web. doi:10.1021/bi602345a.
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Goobes, Rivka, Goobes, Gil, Shaw, Wendy J., Drobny, Gary P., Campbell, Charles T., & Stayton, Patrick. Thermodynamic Roles of Basic Amino Acids in Statherin Recognition of Hydroxyapatite. United States. doi:10.1021/bi602345a.
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Goobes, Rivka, Goobes, Gil, Shaw, Wendy J., Drobny, Gary P., Campbell, Charles T., and Stayton, Patrick. Tue . "Thermodynamic Roles of Basic Amino Acids in Statherin Recognition of Hydroxyapatite". United States. doi:10.1021/bi602345a.
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@article{osti_909672,
title = {Thermodynamic Roles of Basic Amino Acids in Statherin Recognition of Hydroxyapatite},
author = {Goobes, Rivka and Goobes, Gil and Shaw, Wendy J. and Drobny, Gary P. and Campbell, Charles T. and Stayton, Patrick},
abstractNote = {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.},
doi = {10.1021/bi602345a},
journal = {Biochemistry, 46(16):4725-4733},
number = 16,
volume = 46,
place = {United States},
year = {Tue Apr 24 00:00:00 EDT 2007},
month = {Tue Apr 24 00:00:00 EDT 2007}
}
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Journal Article:
DOI:  10.1021/bi602345a
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  • ; ; ; ... - Journal of the American Chemical Society
    Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), Ca10(PO4)6- (OH)2, the main mineral component of bone and teeth. There is remarkably little known about the protein structure-function relationships and the recognition processes governing hard tissue engineering. It is well-known that several salivary proteins (statherin) and peptides (SN-15, N-terminal 15 amino fragment of statherin) bind strongly to HAP to regulate crystal growth.1 In this work, we describe how solid-state NMR can be used to identify which amino acid side chainsmore » of SN-15 (DpSpSEE15NKFLRRIGRFG) interact with the HAP surface, even in the presence of phosphorylated side chains. Prior structural studies have indicated that the second through twelfth amino acids are R-helical in full length statherin on HAP, while the SN-15 fragment is in an extended structure toward the N-terminus, only gaining R-helical structure at the seventh amino acid. Additionally, prior dynamics studies have indicated that the region from the seventh amino acid to the C-terminus interacts less strongly with the HAP surface than the first six amino acids.« less

  • ; ; ; ... - Journal of the American Chemical Society, 128(16):5364-5370
    Extracellular matrix proteins regulate hard tissue growth by acting as adhesion sites for cells, by triggering cell signaling pathways, and by directly regulating the primary and/or secondary crystallization of hydroxyapatite, the mineral component of bone and teeth. Despite the key role that these proteins play in the regulation of hard tissue growth in humans, the exact mechanism used by these proteins to recognize mineral surfaces is poorly understood. Interactions between mineral surfaces and proteins very likely involve specific contacts between the lattice and the protein side chains, so elucidation of the nature of interactions between protein side chains and theirmore » corresponding inorganic mineral surfaces will provide insight into the recognition and regulation of hard tissue growth. Isotropic chemical shifts, chemical shift anisotropies (CSAs), NMR line-width information, 13C rotating frame relaxation measurements, as well as direct detection of correlations between 13C spins on protein side chains and 31P spins in the crystal surface with REDOR NMR show that, in the peptide fragment derived from the N-terminal 15 amino acids of salivary statherin (i.e., SN-15), the side chain of the phenylalanine nearest the C-terminus of the peptide (F14) is dynamically constrained and oriented near the surface, whereas the side chain of the phenylalanine located nearest to the peptide?s N-terminus (F7) is more mobile and is oriented away from the hydroxyapatite surface. The relative dynamics and proximities of F7 and F14 to the surface together with prior data obtained for the side chain of SN-15's unique lysine (i.e., K6) were used to construct a new picture for the structure of the surface-bound peptide and its orientation to the crystal surface.« less

  • ; ; ; ... - Journal of Physical Chemistry B, 110(18):9324-9332
    Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), Ca10(PO4)6(OH)2, the main mineral component of bone and teeth. Key to understanding the structural basis of protein-crystal recognition and protein control of hard tissue growth is the nature of interactions between the protein side chains and the crystal surface. In an earlier work we have measured the proximity of the lysine (K6) side chain in an SN-15 peptide fragment of the salivary protein statherin adsorbed to the Phosphorus-rich surface of HAPmore » using solid-state NMR recoupling experiments. 15N(31P) rotational echo double resonance (REDOR) NMR data on the side-chain nitrogen in K6 gave rise to three different models of protein-surface interaction to explain the experimental data acquired. In this work we extend the analysis of the REDOR data by examining the contribution of interactions between surface phosphorus atoms to the observed 15N REDOR decay. We performed 31P-31P recoupling experiments in HAP and (NH4)2HPO4 (DHP) to explore the nature of dipolar coupled 31P spin networks. These studies indicate that extensive networks of dipolar coupled 31P spins can be represented as stronger effective dipolar couplings, the existence of which must be included in the analysis of REDOR data. We carried out 15N(31P) REDOR in the case of DHP to determine how the size of the dephasing spin network influences the interpretation of the REDOR data. Although use of an extended 31P coupled spin network simulates the REDOR data well, a simplified 31P dephasing system composed of two spins with a larger dipolar coupling also simulates the REDOR data and only perturbs the heteronuclear couplings very slightly. The 31P-31P dipolar couplings between phosphorus nuclei in HAP can be replaced by an effective dipolar interaction of 600 Hz between two 31P spins. We incorporated this coupling and applied the above approach to reanalyze the 15N(31P) REDOR of the lysine side chain approaching the HAP surface and have refined the binding models proposed earlier. We obtain 15N-31P distances between 3.3 and 5 ? from these models that are indicative of the possibility of a lysine-phosphate hydrogen bond.« less

  • ; ; ; ... - Journal of the American Chemical Society

  • ; ; ; ... - Biochemistry, 40(51):15451-15455
    Proteins directly conrol the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface reamain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composite technologies. Here, we have used solid-state NMR techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxypapatite (HAP) surface. Backbone secondary structure for the N-terminal dodecyl region was determined using a combination of homonuclear and heteronuclear dipolar recoupling techniques. Both sets of experiments indicatemore » the N-terminus is a-helical in character with the residues directly binding to the HAP being stabilized in the a-helical conformation by the presecne of water. Dynamic NMR studies demonstrate that the highly anionic N-terminus is strongly adsorbed and immobilized on the HAP surface, while the middle and C-terminal regions of this domain are mobile and thus weakly interacting with the mineral surface. The direct binding footprint of staterin is thus localized to the negatively charged N-terminal pentapeptide sequence. Study of a site-directed mutant demonstrated that alteration of the only anionic side chain ouside of this domain did not affect the dynamics of statherin on the HAP surface, suggesting that it does not play an important role in HAP binding.« less