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Identification and characterization of Fluorescent Protein from marine organisms and potentially applications

von Dr. Tiziana Masullo

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Statistik und Sichtungsnachweis dieser Seite findet sich am Artikelende
[1.] Tim/Fragment 011 01 - Diskussion
Zuletzt bearbeitet: 2014-10-25 07:29:43 Hindemith
BauernOpfer, Fragment, Gesichtet, SMWFragment, Schutzlevel sysop, Tim, Wiedenmann 2000

Typus
BauernOpfer
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 11, Zeilen: 1-28
Quelle: Wiedenmann 2000
Seite(n): 14095, Zeilen: l.col: 1 ff.
The authors (Wiedenmann et al., 2000) noted that the two green fluorescent pigments can be distinguished by their spectral properties in the tentacles of A. sulcata var. rufescens although they could not be separated. This indicates that the two proteins have identical molecular weights or that they reflect two states of the same protein. The second hypothesis is supported by the construction of a cDNA library. In fact they assumed the existence of a second stable conformation of AsGFP499, with red-shifted fluorescence, which can be formed only in the expressing cells of the sea anemone.

Also the orange fluorescent protein asFP595 and the no [sic] fluorescent red protein asCP562 most likely represent two states of the same protein. Both proteins have the same molecular weight. A striking argument for their relationship is the finding that after a denaturation-renaturation process both asFP595 and asCP562 exhibit orange fluorescence with an emission maximum that matches that of asFP595. Therefore, the primary structure of asCP562 also must carry all features necessary for orange fluorescence. The putative semi-β-can structure of asCP562 can give a possible explanation of these phenomena. For GFP fluorescence the β-can structure is essential. They proposed the formation of a β-can-like structure in a multimerization process. This type of β-can consists of at least two molecules of asCP562. Their hypothesis was supported by the finding that both asCP562 and asFP595 show apparent molecular masses of 66 kDa. This molecular mass corresponds exactly to a tetramer consisting of four molecules asCP562 (4*16.5 kDa) (Table 1). Under the conditions necessary for renaturation of asFP595 a second fluorescent band can be observed if the samples are denatured without the presence of β-mercaptoethanol (fig. 4B). The second band migrates at twice the molecular mass of asFP595. This result confirms the hypothesis that asFP595 consists of at least two asCP562 monomers.

Unlike GFP dimers, in asFP595 disulfide bonds seem to be involved in dimer interactions as a complete splitting is only possible under reducing conditions. However, to obtain the fluorescent state, it seems likely that a β-can-like structure is formed in which at least two molecules of asCP562 are linked with a disulfide bond.

Two green fluorescent pigments can be distinguished by their spectral properties in the tentacles of A. sulcata var. rufescens although they could not be separated (Fig. 2A). This indicates that the two proteins have identical molecular weights or that they reflect two states of the same protein. The second hypothesis is supported by the construction of a cDNA library. It resulted in the cloning of several hundred GFPs of the asFP499 type. As the content of asFP499 and asFP522 in the tissue of tentacles is comparably high, one would expect that this ratio should be detectable in the library as well. We therefore assume the existence of a second stable conformation of asFP499, with red-shifted fluorescence, which can be formed only in the expressing cells of the sea anemone.

Also the orange fluorescent protein asFP595 and the non-fluorescent red protein asCP562 most likely represent two states of the same protein. Both proteins have the same molecular weight. A striking argument for their relationship is the finding that after a denaturation-renaturation process both asFP595 and asCP562 exhibit orange fluorescence with an emission maximum that matches that of asFP595. Therefore, the primary structure of asCP562 also must carry all features necessary for orange fluorescence. The putative semi-β-can structure of asCP562 can give a possible explanation of these phenomena. For GFP fluorescence the β-can structure is essential. [...] We propose the formation of a β-can-like structure in a multimerization process. This type of β-can consists of at least two molecules of asCP562. Together they could form a β-can with at least 12 β-strands surrounding at least two stretches homologous to the fluorophore region of GFP. Our hypothesis is supported by the finding that both asCP562 and asFP595 show apparent molecular masses of 66 kDa (Fig. 3.1 and 3.2). This molecular mass corresponds exactly to a tetramer consisting of four molecules asCP562 (4 x 16.5 kDa) (Table 1). Under the conditions necessary for renaturation of asFP595 a second fluorescent band can be observed if the samples are denatured without the presence of β-mercaptoethanol (Fig. 4B) The second band migrates at twice the molecular mass of asFP595. This result confirms the hypothesis that asFP595 consists of at least two asCP562 monomers. Unlike GFP dimers, in asFP595 disulfide bonds seem to be involved in dimer interactions as a complete splitting is only possible under reducing conditions. [...] However, to obtain the fluorescent state, it seems likely that a β-can-like structure is formed in which at least two molecules of asCP562 are linked with a disulfide bond.

Anmerkungen

The source is given at the beginning of the page, but without clear indication that is meant to reference any more than maybe the first paragraph of the page.

Sichter
(SleepyHollow02), Hindemith


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