Model peptide studies of sequence repeats derived from the intracrystalline biomineralization protein, SM50. II. Pro,Asn-Rich tandem repeats

Abstract

In the biomineralization process, a number of Pro‐rich proteins participate in the formation of three‐dimensional supramolecular structures. One such protein superfamily, the Pro,Gly‐rich sea urchin intracrystalline spicule matrix proteins, form protein–protein supramolecular assemblies that modify the microstructure of the inorganic mineral phase (calcite) within embryonic sea urchin spicules and adult sea urchin spines. These proteins represent a useful model for understanding Pro sequence usage and the resulting generation of extended or “open” structures for protein–protein and/or protein–crystal recognition. In the sea urchin spicule matrix protein, SM50 (Strongylocentrotus purpuratus), there exists an unusual 20‐residue Pro,Asn‐containing repeat, PNNPNNPNPNNPNNPNNPNP, which links the upstream 15‐residue C‐terminal domain and the downstream 211‐residue β‐spiral repeat domain. To define the structural preferences of this 20‐residue repeat, we created a 20‐residue N‐ and C‐terminal “capped” peptidomimetic of this sequence. Using far‐uv CD dichroism, CHα and α‐15N conformational shifts, 3JNH‐CHα coupling constants, sequential dNN(i, i + 1) rotating frame nuclear Overhauser effect connectivities, dαN(i, i + 1)/dNN(i, i + 1) intensity ratios, amide temperature shift coefficients, amide solvent exchange, and simulated annealing refinement protocols, we have determined that this 20‐residue repeat motif adopts an extended “twist” structure consisting of turn‐ and coil‐like regions. These findings are consistent with previous studies, which have shown that Pro‐rich tandem repeats adopt extended, flexible structures in solution. We hypothesize that this 20‐residue repeat may fulfill the role of a mineral‐binding domain, a protein–protein docking domain, or as an internal “molecular spacer” for the SM50 protein during spicule biocomposite formation. © 2000 John Wiley & Sons, Inc. Biopoly 54: 464–475, 2000

Intrinsically Disordered Proteins in Biomineralization

“…Secondly, PNNP repeat motifs are believed to play an important role in mineralization. P-rich sequences often exhibit the extended, flexible structure responsible for protein-protein and/or protein-crystal recognition, while N residues represent putative sites for side chain hydrogen bonding (Zhang et al, 2000). PNNP repeats adopt an extended, twisted conformation consisting of a mixture of turn-like and coil-like regions, most likely interconverting with the coiled-coil state (Zhang et al, 2000).…”

“…P-rich sequences often exhibit the extended, flexible structure responsible for protein-protein and/or protein-crystal recognition, while N residues represent putative sites for side chain hydrogen bonding (Zhang et al, 2000). PNNP repeats adopt an extended, twisted conformation consisting of a mixture of turn-like and coil-like regions, most likely interconverting with the coiled-coil state (Zhang et al, 2000). Several possible roles for PNNP have been suggested: (i) as a mineral recognition domain, (ii) as a self-assembly domain, (iii) as a molecular spacer (Zhang et al, 2000).…”

Expressed sequence tag profiles from calcifying and non-calcifying cultures of Emiliania huxleyi

“…Whether or not protein-coding genes play a role in regulating the growth, shape, and physical properties of the coccoliths, as they do in biomineralization processes in the diatom, oyster, sea urchin, and mollusk (Gotliv et al 2003;Hazelaar et al 2003;Illies et al 2002;Miyamoto et al 1996;Zhang et al 2000) is not known. Nineteen of the most abundant ESTs in the f/50 library derived from lith forming cells show no significant match to any sequence present in the f/2 library derived from non-calcifying cells, nor do they show any significant match to any of any GenBank sequences, including biomineralization proteins.…”

“…Nineteen of the most abundant ESTs in the f/50 library derived from lith forming cells show no significant match to any sequence present in the f/2 library derived from non-calcifying cells, nor do they show any significant match to any of any GenBank sequences, including biomineralization proteins. Our laboratory intends to obtain full length sequences for these clones that may represent marker sequences for calcification, and characterize their biochemical and biophysical properties with respect to what is known regarding silaffins (Kroger et al 2000;Kroger et al 2001;Kroger et al 2002), lustrin , pearlin (Miyashita et al 2000), nacrein (Miyamoto et al 1996) and other biomineralization proteins (Michenfelder et al 2003;Wustman et al 2002;Zhang et al 2000). Many biomineral-associated proteins tend to have no distinct secondary or tertiary structure but rather feature extended, repeating b-turn, loop, or random coil conformations (Michenfelder et al 2003;Wustman et al 2002;Xu and Evans 1999;Zhang et al 2002;Zhang et al 2000) and contain specific amino acid sequence motifs.…”

The multiple effects of amino acids on the early stages of calcium carbonate crystallization

“…Several groups have examined short peptides containing repeats of proline and glycine on mineralization [29,31]. Others have used short peptides with consensus groups from specific known mineralization proteins [18,26,27,32].…”