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

von Dr. Tiziana Masullo

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[1.] Tim/Fragment 014 05 - Diskussion
Zuletzt bearbeitet: 2014-10-27 04:06:01 Hindemith
BauernOpfer, Fragment, Gesichtet, Lukyanov 2000, SMWFragment, Schutzlevel sysop, Tim

Typus
BauernOpfer
Bearbeiter
SleepyHollow02
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 14, Zeilen: 5-9
Quelle: Lukyanov 2000
Seite(n): 25880, Zeilen: l.col.: 21 ff.
With the exception of a slight shoulder at 530 nm, the protein displays a single absorption wavelength maximum, which occurs at 572 nm (Ԑ572 = 56,200 M-1 cm-1) (fig. 6). As is evident asFP595 absorbs efficiently in the middle range of the visible spectrum but remains translucent when excited with blue or long wavelength red light. Consequently, to the observer, the protein appears intensely purple.

[asFP595 can be transferred by green light from a nonfluorescent off into a fluorescent on state from which it reverts back eventually, but this transition can also be promptly stimulated by gentle irradiation with blue light (Lukyanov et al., 2000).]

With the exception of a slight shoulder at 530 nm, the protein displays a single absorption wavelength maximum, which occurs at 572 nm (e572 = 56,200 M-1 cm-1). As is evident from this analysis, asFP595 absorbs efficiently in the middle range of the visible spectrum but remains translucent when excited with blue or long wavelength red light. Consequently, to the observer, the protein appears intensely purple.
Anmerkungen

The source is mentioned at the end of the next sentence in the next paragraph, which, however, has been taken from another source: Tim/Fragment 014 10

Sichter
(SleepyHollow02), Hindemith

[2.] Tim/Fragment 014 10 - Diskussion
Zuletzt bearbeitet: 2014-11-27 22:14:59 Hindemith
Andresen et al 2005, Fragment, Gesichtet, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Tim

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Hindemith
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Untersuchte Arbeit:
Seite: 14, Zeilen: 10-15
Quelle: Andresen et al 2005
Seite(n): 13070, Zeilen: 44 ff.
asFP595 can be transferred by green light from a nonfluorescent off into a fluorescent on state from which it reverts back eventually, but this transition can also be promptly stimulated by gentle irradiation with blue light (Lukyanov et al., 2000). The "on-off" cycle can be repeated many times. However, with its low quantum yield (˂0.001), and comparatively slow switching kinetics, the photochromic properties of asFP595 are far from being optimal. It can be transferred by green light from a nonfluorescent off into a fluorescent on state from which it reverts back eventually, but this transition can also be promptly stimulated by gentle irradiation with blue light (6). The "on-off" cycle can be repeated many times. However, with its low quantum yield (<0.001, ref. 6) and comparatively slow switching kinetics, the photochromic properties of asFP595 are far from being optimal.

6. Lukyanov, K. A., Fradkov, A. F., Gurskaya, N. G., Matz, M. V., Labas, Y. A., Savitsky, A. P., Markelov, M. L., Zaraisky, A. G., Zhao, X. N., Fang, Y., et al. (2000) J. Biol. Chem. 275, 25879–25882.

Anmerkungen

The source is only mentioned in the next paragraph.

Sichter
(Hindemith), SleepyHollow02

[3.] Tim/Fragment 014 19 - Diskussion
Zuletzt bearbeitet: 2014-11-27 22:15:03 Hindemith
Andresen et al 2005, BauernOpfer, Fragment, Gesichtet, SMWFragment, Schutzlevel sysop, Tim

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Hindemith
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Untersuchte Arbeit:
Seite: 14, Zeilen: 19-24, 27-33
Quelle: Andresen et al 2005
Seite(n): 13071, Zeilen: r.col. 14 ff.
The quantum yield of these variants was major as compared with that of the wt asFP595 protein. As in all GFP-like proteins, the asFP595 chromophore resides in a helical segment, which is enclosed in an 11-stranded β-barrel (Ormo et al., 1996; Wall et al., 2000; Yang et al., 1996). The tripeptide M63-Y64-G65 rearranges to a chromophoric, conjugated 2-iminomethyl-5-(4-hydroxybenzylidene) imidazolinone (MYG) system.

[...]

Unlike most other GFP-like proteins, the protein backbone is broken between C62 and the chromophore. Nevertheless, the former M63 Cα and backbone nitrogen atoms are in plane with the imidazolinone ring and, thus, part of the conjugated system. The imino group expands the conjugated system of MYG, likely accounting for the shift of the absorption maximum toward a longer wavelength (572 nm) as compared with that of GFP (470 nm). The MYG chromophore of the off state asFP595 exclusively adopts the trans conformation.

As in all GFP-like proteins, the asFP595 chromophore resides in a helical segment, which is enclosed in an 11-stranded β-barrel (20–23). The tripeptide M63-Y64-G65 rearranges to a chromophoric, conjugated 2-iminomethyl-5-(4-hydroxybenzylidene) imidazolinone (MYG) system, in agreement with previous biochemical analysis of proteolytic peptides (24). Unlike most other GFP-like proteins, the protein backbone is broken between C62 and the chromophore. Nevertheless, the former M63 Cα and backbone nitrogen atoms are in plane with the imidazolinone ring and, thus, part of the conjugated system. The imino group expands the conjugated system of MYG, likely accounting for the shift of the absorption maximum toward a longer wavelength (572 nm) as compared with that of GFP (470 nm). The MYG chromophore of the off state asFP595 exclusively adopts the trans conformation.

[...]

[...] This variant has 12 times the fluorescence quantum yield as compared with that of the wt asFP595 protein (6).


6. Lukyanov, K. A., Fradkov, A. F., Gurskaya, N. G., Matz, M. V., Labas, Y. A., Savitsky, A. P., Markelov, M. L., Zaraisky, A. G., Zhao, X. N., Fang, Y., et al. (2000) J. Biol. Chem. 275, 25879–25882.

20. Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y. & Remington, S. J. (1996) Science 273, 1392–1395.

21. Wall, M. A., Socolich, M. & Ranganathan, R. (2000) Nat. Struct. Biol. 7, 1133–1138.

22. Yang, F., Moss, L. G. & Phillips, G. N., Jr. (1996) Nat. Biotechnol. 14, 1246–1251.

23. Yarbrough, D., Wachter, R. M., Kallio, K., Matz, M. V. & Remington, S. J. (2001) Proc. Natl. Acad. Sci. USA. 98, 462–467.

24. Zagranichny, V. E., Rudenko, N. V., Gorokhovatsky, A. Y., Zakharov, M. V., Balashova, T. A. & Arseniev, A. S. (2004) Biochemistry 43, 13598– 13603.

Anmerkungen

The source is mentioned right before the documented passage. One could assume that the first documented sentence describes the results of Andresen et al. (2005), what follows, however, is attributed to other sources.

Sichter
(Hindemith), SleepyHollow02


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