Amelogenin Secondary, Tertiary, and Guaternary Structure with NMR and AFM

Objectives: Protein structure (secondary, tertiary, and quaternary) is proposed to play an important role in biomineralization in general and in the function of amelogenin in enamel formation in particular. The wildtype protein has a lack of sescondary or tertiary structure in solution, but becomes structured and less flexible when bound to HAP. The quaternary structure upon binding to HAP is also signficantly different than in solution. Point mutations of amelogenin cause amelogenesis imperfecta. The goal of these studies is to understand the secondary, tertiary, and quaternary structures of two naturally occurring mutants, T21I and P41T, to correlate structure and function in enamel development.
Methods: Using solution and solid state NMR and AFM, the secondary, tertiary, and quaternary structure of full-length wildtype amelogenin and two naturally occurring amelogenin mutants will be compared in solution and bound to HAP.
Results: The structural flexibility and quaternary structure is altered in solution and on HAP as a function of mutation, while minimal changes in secondary structure are observed. Trypsin and MMP-20 digestion suggest hindered accessibility to the digestion sites for the T21I mutation.
Conclusions: The differences as a function of single site mutation suggest that these mutations are critical in controlling structure, and/or protein-protein or protein-HAP interactions. Our results suggest that increases in flexibility may be controlling hindered cleavage by either trypsin or MMP-20.