Liu, Liyun, Im, H., Cegielski, M., LeMagueres, P., Schultz,W., Krause, K., and Hastings, J.W. (2003) Characterization and Crystallization of Active Domains of a Novel Luciferase from a Marine Dinoflagellate. Acta Cryst. D59: 761-764
ABSTRACT
Characterization and crystallization of active domains of a novel luciferase from a marine dinoflagellate Lingulodinium polyedrum luciferase is a bioluminescent protein found in the marine dinoflagellate formerly known as Gonyaulax. It is located in organelles called scintillons that emit brief and bright flashes of light that are regulated by an endogenous circadian clock. The complete luciferase molecule has a molecular mass of 136 994 Da and contains three homologous domains, each of which is a separately active luciferase. Two of these domains, D2-LCF and D3-LCF, have been cloned, expressed and crystallized. Crystals of D2-LCF were obtained from PEG 10 000 in space group P212121, with unit-cell parameters a = 49.1, b = 104.7, c = 180.3 Å. They diffract to 2.9 Å on a rotating anode. Crystals of D3-LCF were grown from PEG 2000 in space group P212121, with unit-cell parameters a = 58.86, b = 63.98, c = 95.76 Å. They diffract to 2.3 Å on a rotating anode. 420. Vectorial Labeling of Dinoflagellate Cell Surface Proteins The objective of the current study was to identify cell surface proteins in the dinoflagellate Lingulodinium polyedrum. Proteins on the surface of living cells that had regions exposed to the external medium were labeled with Na125I. Following partial purification of membrane proteins and analysis by two-dimensional gel electrophoresis and autoradiography, a protein of roughly 43 kDa was found to have incorporated the radiolabel. This protein was cloned using a combination of protein microsequencing and PCR amplification. The derived protein sequence in the cDNA has a signal peptide at the N-terminal end of the protein, and thus represents the first plasma membrane protein ever reported for a dinoflagellate. The function of the protein is unknown, but its cloning provides a proof of principle for the general use of vectorial labeling in identifying cell surface proteins of marine algae.