The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution

Tarasevich, Barbara J.; Philo, John S.; Maluf, Nasib Karl; Krueger, Susan; Buchko, Garry W.; Lin, Genyao; Shaw, Wendy J.

doi:10.1016/j.jsb.2014.10.007

The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution

Journal Article · Sat Oct 25 00:00:00 EDT 2014 · Journal of Structural Biology

DOI:https://doi.org/10.1016/j.jsb.2014.10.007· OSTI ID:1182923

Tarasevich, Barbara J. ^[1]; Philo, John S. ^[2]; Maluf, Nasib Karl ^[2]; Krueger, Susan ^[3]; Buchko, Garry W. ^[1]; Lin, Genyao ^[4]; Shaw, Wendy J. ^[1]

Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Alliance Protein Lab., Inc., San Diego, CA (United States)
National Inst. of Standards and Technology (NIST), Gaithersburg, MD (United States)
WSP Chemicals & Technology, LLC, Leetsdale, PA (United States)

Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in promoting nucleation, controlling growth, and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, the quaternary structure of LRAP is not as well studied. Here, analytical ultracentrifugation sedimentation velocity (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP over a range of pH values, ionic strengths, and concentrations. SV has advantages over other techniques in accurately quantifying protein speciation in polydisperse solutions. We found that the monomer was the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7 to 4.1, pH 4.5 to 8, 50 mmol/L( mM) to 200 mM NaCl, 0.065 to 2 mg/mL). The monomer was also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregated in a narrow pH range near the isoelectric point (pH 4.1). We conclude that LRAP does not form nanospheres under physiological solution conditions. Both SV and SANS showed that the LRAP monomer has a radius of ~2.0 nm and adopts an extended structure which solution NMR studies show is intrinsically disordered. This work provides new insights into the tertiary and quaternary structure of LRAP and further evidence that the monomeric species is an important functional form of amelogenins

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1182923

Alternate ID(s):: OSTI ID: 1359491

Report Number(s):: PNNL-SA--105350; 44691; 41891; 48235; 47735; 400412000

Journal Information:: Journal of Structural Biology, Journal Name: Journal of Structural Biology Journal Issue: 1 Vol. 190; ISSN 1047-8477

Publisher:: ElseiverCopyright Statement

Country of Publication:: United States

Language:: English

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