PHOSPHOPHORYN, A BIOMINERALIZATION TEMPLATE PROTEIN - PH-DEPENDENT PROTEIN-FOLDING EXPERIMENTS

The protein folding behavior of a polyelectrolyte protein, bovine dentine phosphophoryn (BDPP), in the pH range of 1.82-11.0 has been investigated. One- and two-dimensional nmr spectroscopy has been utilized to obtain proton spin assignments for amino acid residues in D2O and in H2O. One-dimensional P-31-nmr experiments verify the existence of three separate classes of O-phosphoserine (PSer) resonances in BDPP (alpha,beta,chi), representing three distinct PSer residue populations at pH 6.94. By means of pH titration and H-1-nmr, five populations of Asp residues can be identified. Three of these populations exhibit secondary inflection points on their pH titration curves that correspond to an observed pK(a) of 6.17-6.95. The presence or absence of secondary inflection points for Asp populations and the P-31-nmr chemical shift dispersion for the three PSer residue populations indicate that BDPP may be comprised of homologous (Asp-Asp)(n). (PSer-PSer)(n), and heterologous (PSer-Asp)(n) sequences arranged into polyelectrolyte cluster regions. The pH titration also revealed that certain populations of Ser, Gly, and Pro residues in BDPP exhibit pH-dependent resonance frequency shifts. The ''apparent'' pK(a) for the transition points of these frequency shifts corresponds to either the pK(a)(1) of Pser monophosphate ester and/or the pK(a) of Asp COOH group of BDPP polyelectrolyte regions. On the basis of these transition points, we can assign four types of Ser, Gly, or Pro-containing ''intervening'' regions in BDPP, based on their sensitivity to protonation and deprotonation events occurring at (Asp)(n), (PSer)(n), or (PSer-Asp)(n) anionic cluster regions that flank the intervening regions. Our H-1-nmr experiments also reveal that BDPP assumes a folded conformation at low pH. As the pH increases, this conformation undergoes several unfolding transitions as the BDPP molecule assumes more open conformations in response to increased electrostatic repulsion between polyelectrolyte anionic regions in the protein. These folding-unfolding transitions are mediated by the intervening regions, which act as ''hinges'' to allow the polyelectrolyte regions to fold relative to one another. (C) 1994 John Wiley & Sons, Inc.