title: Neutron reflectometry studies of the adsorbed structure of the amelogenin, LRAP.
abstract: Amelogenins make up over 90% of the protein present during enamel formation and have been demonstrated to be critical in proper enamel development, but the mechanism governing this control is not well understood. Leucine-rich amelogenin peptide (LRAP) is a 59-residue splice variant of amelogenin and contains the charged regions from the full protein thought to control crystal regulation. In this work, we utilized neutron reflectivity (NR) to investigate the structure and orientation of LRAP adsorbed from solutions onto molecularly smooth COOH-terminated self-assembled monolayer (SAM) surfaces. Sedimentation velocity (SV) experiments revealed that LRAP is primarily a monomer in saturated calcium phosphate (SCP) solutions (0.15 M NaCl) at pH 7.4. LRAP adsorbed as ∼32 Å thick layers at ∼70% coverage as determined by NR. Rosetta simulations of the dimensions of LRAP in solution (37 Å diameter) indicate that the NR determined z dimension is consistent with an LRAP monomer. SV experiments and Rosetta simulations show that the LRAP monomer has an extended, asymmetric shape in solution. The NR data suggests that the protein is not completely extended on the surface, having some degree of structure away from the surface. A protein orientation with the C-terminal and inner N-terminal regions (residues ∼8-24) located near the surface is consistent with the higher scattering length density (SLD) found near the surface by NR. This work presents new information on the tertiary and quaternary structure of LRAP in solution and adsorbed onto surfaces. It also presents further evidence that the monomeric species may be an important functional form of amelogenin proteins.
authors.affiliation: Pacific Northwest National Laboratory, Richland, Washington 99354, United States. barbara.tarasevich@pnnl.gov
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Tarasevich
authors.forename: Ursula
authors.initials: U
authors.lastname: Perez-Salas
authors.forename: David L
authors.initials: DL
authors.lastname: Masica
authors.forename: John
authors.initials: J
authors.lastname: Philo
authors.forename: Paul
authors.initials: P
authors.lastname: Kienzle
authors.forename: Susan
authors.initials: S
authors.lastname: Krueger
authors.forename: Charles F
authors.initials: CF
authors.lastname: Majkrzak
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Gray
authors.forename: Wendy J
authors.initials: WJ
authors.lastname: Shaw
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D002130:Calcium Phosphates / Q000737:chemistry; D003746:Dental Enamel Proteins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009502:Neutrons; D017434:Protein Structure, Tertiary; D012031:Refractometry; D013499:Surface Properties
chemical_list: D002130:Calcium Phosphates; D003746:Dental Enamel Proteins; C067432:leucine-rich amelogenin peptide; C020243:calcium phosphate