Molecular characterization of the 30-AA N-terminal mineral interaction domain of the biomineralization protein AP7.
The AP7 protein is one of several mollusk shell proteins which are responsible for aragonite polymorph formation and stabilization within the nacre layer of the Pacific red abalone, H. rufescens. Previously, we demonstrated that the 30-AA N-terminal domain of AP7, denoted as AP7-1, exists as an unfolded sequence and possesses the capability of inhibiting calcium carbonate crystal growth in vitro via growth step frustration or interruption. However, very little is known with regard to the interactive capabilities of this sequence with Ca(II) and with calcium carbonates. Using multidisciplinary techniques, we determine that the AP7-1 polypeptide interacts with Ca(II) ions at the -DD- sequence clusters, yet retains its unfolded, conformationally labile structure in the presence of Ca(II) ions. Further, NMR experiments reveal that the extended structured sequence blocks, -GNGM-, -SVRTQG-, and -ISYL, exhibit motional, chemical exchange, and/or backbone geometry perturbations in response to Ca(II) interactions with AP7-1. Solid-state NMR magic angle spinning studies verify that during the course of in vitro calcium carbonate crystal growth, AP7-1 becomes bound to calcite fragments and cannot be entirely displaced from the mineral fragments using competitive Ca(II) washing. Finally, using a scrambled sequence version of the AP7-1 polypeptide, we observe that sequence scrambling does not adversely affect the crystal growth inhibitory activity of AP7-1, suggesting that the amino acid composition of AP7-1 may be more critical to growth step inhibition than the linear ordering of amino acids.
Kim
Il Won
IW
Center for Biomolecular Materials Spectroscopy, Laboratory for Chemical Physics, New York University, 345 East 24th Street, New York, New York 10010, USA.
Morse
Daniel E
DE
Evans
John Spencer
JS
D000595:Amino Acid Sequence; D000818:Animals; D002113:Calcification, Physiologic; D002118:Calcium / Q000737:chemistry*; D002942:Circular Dichroism; D007477:Ions / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D008974:Mollusca / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*
D007477:Ions; D010446:Peptide Fragments; D011506:Proteins; D002118:Calcium