Perlinhibin, a cysteine-, histidine-, and arginine-rich miniprotein from abalone (Haliotis laevigata) nacre, inhibits in vitro calcium carbonate crystallization

We have isolated a 4.785 Da protein from the nacreous layer of the sea snail Haliotis laevigata ( greenlip abalone) shell after demineralization with acetic acid. The sequence of 41 amino acids was determined by Edman degradation supported by mass spectrometry. The most abundant amino acids were cysteine ( 19.5%), histidine ( 17%), and arginine ( 14.6%). The positively charged amino acids were almost counterbalanced by negatively charged ones resulting in a calculated isoelectric point of 7.86. Atomic- force microscopy studies of the interaction of the protein with calcite surfaces in supersaturated calcium carbonate solution or calcium chloride solution showed that the protein bound specifically to calcite steps, inhibiting further crystal growth at these sites in carbonate solution and preventing crystal dissolution when carbonate was substituted with chloride. Therefore this protein was named perlinhibin. X- ray diffraction investigation of the crystal after atomic- force microscopy growth experiments showed that the formation of aragonite was induced on the calcite substrate around holes caused by perlinhibin crystal- growth inhibition. The strong interaction of the protein with calcium carbonate was also shown by vapor diffusion crystallization. In the presence of the protein, the crystal surfaces were covered with holes due to protein binding and local inhibition of crystal growth. In addition to perlinhibin, we isolated and sequenced a perlinhibin- related protein, indicating that perlinhibin may be a member of a family of closely related proteins.