DELINEATION OF CONFORMATIONAL PREFERENCES IN HUMAN SALIVARY STATHERINBY H-1, P-31 NMR AND CD STUDIES - SEQUENTIAL ASSIGNMENT AND STRUCTURE-FUNCTION CORRELATIONS

Membrane-induced solution structure of human salivary statherin, a 43 amino acid residue acidic phosphoprotein, has been investigated by two -dimensional proton nuclear magnetic resonance (2D H-1 NMR) spectrosco py. NMR assignments and structural analysis of this phosphoprotein was accomplished by analyzing the pattern of sequential and medium range NOEs, alpha CH chemical shift perturbations and deuterium exchange mea surements of the amide proton resonances. The NMR data revealed three distinct structural motifs in the molecule: (1) an alpha-helical struc ture at the N-terminal domain comprising Asp(1)-Tyr(16), (2) a polypro line type II (PPII) conformation predominantly occurring at the middle proline-rich domain spanning Gly(19)-Gln(35), and (3) a 3(10)-helical structure at the C-terminal Pro(36)-Phe(43) sequence. Presence of a f ew weak d(alpha N(i, i+2)) NOEs suggests that N-terminus also possesse s ;minor population of 3(10)-helical conformation. Of the three second ary structural elements, helical structure formed by the N-terminal re sidues, Asp(1)-Ile(11) appears to be more rigid as observed by the rel atively very slow exchange of amide hydrogens of Glu(5)-Ile(11). P-31 NMR experiments clearly indicated that N-terminal domain of statherin exists mainly in disordered state in water whereas, upon addition of s tructure stabilizing co-solvent, 2,2,2-trifluorethanol (TFE), it showe d a strong propensity for helical conformation. Calcium ion interactio n studies suggested that the disordered N-terminal region encompassing the two vicinal phosphoserines is essential for the binding of calciu m ions in vivo. Results from the circular dichroism (CD) experiments w ere found to be consistent with and complimentary to the NMR data and provided an evidence that non-aqueous environment such as TFE, could i nduce the protein to fold into helical conformation. The findings that the statherin possesses blended solvent sensitive secondary structura l elements and the requirement of non-structured N-terminal region und er aqueous environment in calcium ion interaction may be invaluable to understand various physiological functions of statherin in the oral f luid.