A solid state NMR dipolar recoupling study of surface interactions of a N-terminal statherin fragment bound to hydroxyapatite

This thesis describes work in solid-state NMR methods development and the application of solid-state NMR techniques to a biological system. Studies comparing homonuclear dipolar recoupling pulse sequences and studies of interactions between biomineralization peptides and inorganic surfaces are presented. There are numerous homonuclear recoupling techniques measuring dipolar couplings from which internuclear distances and torsion angle constraints can be extracted; however, each pulse sequence has its strengths and weaknesses. POST-C7, SPC5, DQDRAWS, SR22, and R14, were characterized as a function of chemical shift anisotropy (CSA) and internuclear distance using six doubly 13 C labeled model compounds. To determine how homonuclear pulse sequences perform under various spin system conditions, the double quantum excitation efficiencies were compared. DQDRAWS and SR22 both excited double quantum with a high efficiency for compounds with long internuclear distances and large CSAs, conditions typical for peptides. These two pulse sequences can be applied to biological systems. Both DQDRAWS and 2-dimensional DQDRAWS were implemented on a 15 amino acid long N-terminal fragment of statherin (SN15), with doubly 13C labeled carbonyls in positions F 7L8. Statherin is a peptide responsible for biomineralization and binds to an inorganic crystal hydroxyapatite (HAP), preventing both nucleation and growth. The torsion angles (&phis;, psi) were determined to be (-58°, -29°), respectively, consistent with an a-helical structure of the unbound peptide. A new method for applying 2D-DQDRAWS over a reduced time was shown to have results with a broader minimum indicating that more data points need to be taken in order to get an accurate answer. 2D-DQDRAWS under challenging signal to noise samples like biological ones is difficult. The interaction of SN15 [ring-13 C6] phenylalanine sidechains with phosphorus in HAP was explored by using 13C T1rho, 1H T 1rho and 13C- 31P REDOR experiments. SN15 F7 was found to be more dynamic than SN15 F14. SN15 F14 also showed REDOR dephasing when bound to HAP while SN15 F7 did not, suggesting that the F14 sidechain is closer to the surface than F7. A model of the orientation of SN15 to HAP was proposed where SN15 loses its helical structure at the C-terminus, allowing F14 to point toward the HAP surface with F7 pointing away.