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Structure of the Ca2+-regulated photoprotein obelin at 1.7 A resolution determined directly from its sulfur substructure.

ABSTRACT: The crystal structure of the photoprotein obelin (22.2 kDa) from Obelia longissima has been determined and refined to 1.7 A resolution. Contrary to the prediction of a peroxide, the noncovalently bound substrate, coelenterazine, has only a single oxygen atom bound at the C2-position. The protein-coelenterazine 2-oxy complex observed in the crystals is photo-active because, in the presence of calcium ion, bioluminescence emission within the crystal is observed. This structure represents only the second de novo protein structure determined using the anomalous scattering signal of the sulfur substructure in the crystal. The method used here is theoretically different from that used for crambin in 1981 (4.72 kDa) and represents a significant advancement in protein crystal structure determination.

SUBMITTER: Liu ZJ

PROVIDER: S-EPMC2144499 | biostudies-other | 2000 Nov

REPOSITORIES: biostudies-other

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Structure of the Ca2+-regulated photoprotein obelin at 1.7 A resolution determined directly from its sulfur substructure.

Liu Z J ZJ Vysotski E S ES Chen C J CJ Rose J P JP Lee J J Wang B C BC

Protein science : a publication of the Protein Society 20001101 11

The crystal structure of the photoprotein obelin (22.2 kDa) from Obelia longissima has been determined and refined to 1.7 A resolution. Contrary to the prediction of a peroxide, the noncovalently bound substrate, coelenterazine, has only a single oxygen atom bound at the C2-position. The protein-coelenterazine 2-oxy complex observed in the crystals is photo-active because, in the presence of calcium ion, bioluminescence emission within the crystal is observed. This structure represents only the ...[more]

PMID: 11152120

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Project description:1. The uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes was investigated with the aim of producing liposome-cell fusion, enabling obelin to be introduced into the cytoplasm of intact cells. 2. Incubation of liposomes containing obelin with rat isolated adipocytes resulted in a time-dependent uptake of entrapped obelin by the adipocytes. The uptake by adipocytes (at 2h) of liposomes prepared from phosphatidylcholine, phosphatidylcholine+phosphatidylserine (molar ratio 4:1) and phosphatidylcholine+N-octadecylamine (molar ratio 4:1) was approx. 6, 10 and 10% of original entrapped obelin per g dry wt. of adipocytes respectively. 3. During incubation with adipocytes some of the liposomes became permeable to Ca(2+) ions, resulting in stimulation of obelin luminescence from within the liposomes. This increase in permeability to Ca(2+) seemed to be the result of the interaction of liposomes with the cell membrane. 4. Approx. 50% of liposome uptake could be inhibited by cytochalasin B (500mum). This was consistent with this uptake being the result of endocytosis. The remaining uptake was probably the result of adhesion of liposomes to the cell membrane. 5. In an attempt to detect the presence of cytoplasmic obelin, after incubation of adipocytes with liposomes, a method of causing a rapid rise in cell-membrane permeability to Ca(2+) was developed in which an anti-cell anti-body-complement reaction occurred at the cell membrane. There was no detectable transfer of active obelin into the cell cytoplasm. 6. After incubation of liposomes with adipocytes in the absence of bovine serum albumin, obelin luminescence from a small proportion of liposomes increased (approx. 1.5%) in response to anti-(5'-nucleotidase) antibody plus complement. 7. It was concluded that under the conditions of these experiments, (a) no detectable transfer (<0.1%) of liposome-entrapped obelin to the adipocyte cytoplasm had occurred, (b) an increase in liposome permeability to Ca(2+) occurred during incubation with adipocytes, (c) at least 50% of liposome uptake by adipocytes was the result of endocytosis, presumably into secondary lysosomes, and the remaining uptake was apparently the result of loose attachment of liposomes to the cell surface, and (d) in the absence of bovine serum albumin, a portion of at least one surface antigen, the ectoenzyme 5'-nucleotidase, was transferred from the adipocyte membrane to the liposome membrane.

Dataset Information

Structure of the Ca2+-regulated photoprotein obelin at 1.7 A resolution determined directly from its sulfur substructure.

Publications

Structure of the Ca2+-regulated photoprotein obelin at 1.7 A resolution determined directly from its sulfur substructure.

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