The Leucine-Rich Amelogenin Peptide (LRAP) is a Partially Structured Monomer in Solution
Barbara J. Tarasevich, John S. Philo, Nasib Karl Mulof, Susan T. Krueger, Garry W. Buchko, Genyao Lin, Wendy J. Shaw
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 peptide (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, 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 multimodal suspensions. We found that the monomer was the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 3.0 to pH 3.9, pH 4.5 to pH 8, 50 mmol/L (mM) to 200 mM NaCl, 0.065 mg/mL 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 agglomerated in a narrow pH range near the isoelectric point (pH 4.1). We conclude that LRAP does not self-assemble to form nanospheres under physiological solution conditions. SV and SANS showed that LRAP has a radius of gyration of approximately equal} 2.0 nm and an extended structure. The size and shape of the protein is the same at pH 3 and pH 7.4, as determined by SANS. This work provides new insights into the tertiary and quaternary structure
, Philo, J.
, Mulof, N.
, Krueger, S.
, Buchko, G.
, Lin, G.
and Shaw, W.
The Leucine-Rich Amelogenin Peptide (LRAP) is a Partially Structured Monomer in Solution, Journal of Structural Biology, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=915738
(Accessed November 29, 2023)