NMR and computational studies on the conformational folding of the biomineralization template, phosphophoryn

Citation

Evans, John Spencer (1993) NMR and computational studies on the conformational folding of the biomineralization template, phosphophoryn. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ky1z-w552. https://resolver.caltech.edu/CaltechTHESIS:10212009-152426877

Abstract

Bovine Dentine Phosphophoryn (BDPP) is a member of the "Asp-rich" superfamily of template macromolecules known as polyelectrolyte mineral matrix proteins (PMMPs). Using 1-D and 2-D NMR multinuclear spectroscopy, protein sequencing, solid-phase peptide synthesis, and molecular modeling, we investigated the capacity of BDPP to fold in solution under certain conditions. It is believed that the folding properties of a template macromolcule are important in the formation of n inorganic mineral phase. In this report, we have established the following regarding BDPP structure: I. BDPP sequence organization can be conceptualized as three types of domains: polyelectrolyte calcium binding domains (PCBD), hinge domains (HD), and the hydrophobic domains (HC). The PCBD regions can be further subdivided into (Asp)_n, (PSer)_n, and (PSerAsp)_n-containing homopolymer and heteropolymer sequence stretches. The HD contain predominantly neutral or uncharged amino acids such as Ser, Gly, and Pro. These HD domain sequences are flanked on either side by PCBD regions. II. BDPP contains a number of Lys sidechains (44/1000 residues) which are believed to form ion pairs with either Asp, Glu, or PSer residues in the protein. The function of this salt-bridging is not understood at this time, but it may be responsible for maintaining the protein template molecule in a conformation that retains a high negative charge density. III. At low pH, or in the presence of divalent cations, BDPP assumes a global conformation that is condensed in particle size. At neutral pH under conditions of low ionic strength and in the absence of divalent cations, this global conformation converts to an extended form. The folding transition between these two conformers is mediated by conformational change in the BD regions which are flanked by specific PCBD sequence regions. IV. The PCBD regions possess some degree of tertiary and secondary structural organization in the absence of divalent cations at low ionic strength. This folding permits the surface charge density of these regions to remain high, relative to that of a random coil conformer. V. BDPP exhibits a selectivity in terms of divalent cation binding sites. Under conditions of low ionic strength and divalent cation depletion, the addition of Cd (II) to BDPP leads to binding at various PCBD sequence stretches, according to the following order: (PSer)_n > (PserAsp)_n > (Asp)_n VI. Modeling studies conducted on PCBD sequence peptides [(Asp)_(20), (PSer)_(20), and (PSerAsp)_(10)] indicate that there is a sequence preference for certain conformers in the presence of Na^+, i.e., (Asp)_(20) forms "supercoils", (PSer)_(20) forms "hairpins", and (PSerAsp)_(10) forms "spirals" or "distorted" hairpins. Each of these conformers features some degree of sidechain folding and/or peptide backbone secondary structure, in support of Mann's hypothesis, as well as the experimental data obtained for BDPP. VII. A peptide mimetic which represents a PCBD-Hinge-PCBD motif of rat α-phosphophoryn, was constructed using FMOC solid-phase peptide synthesis. Using 2-D NMR spectroscopy and monitoring α-CH and sidechain β-CH_2 proton chemical shifts, we can demonstrate that this peptide mimetic folds or collapses under conditions of low pH and divalent cation addition, in a manner similar to that observed for intact BDPP.

Thesis Files