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Angaben zur Quelle [Bearbeiten]

Autor     Karin Nienhaus, Fabiana Renzi, Beatrice Vallone, Jörg Wiedenmann, and G. Ulrich Nienhaus
Titel    Chromophore-Protein Interactions in the Anthozoan Green Fluorescent Protein asFP499
Zeitschrift    Biophysical Journal
Datum    December 2006
Seiten    4210–4220
URL    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635683/pdf/4210.pdf

Literaturverz.   

yes
Fußnoten    yes
Fragmente    5


Fragmente der Quelle:
[1.] Tim/Fragment 006 01 - Diskussion
Zuletzt bearbeitet: 2014-10-15 18:51:06 Singulus
BauernOpfer, Fragment, Gesichtet, Nienhaus 2006, SMWFragment, Schutzlevel sysop, Tim

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[Nienhaus et al. (2006) described the quaternary structure and found that the asymmetric unit of AsGFP contains two identical tetramers related by a no crystallographic symmetry. They are arranged as dimers of dimers, so that two types of subunit interfaces can be distinguished; they are denoted as antiparallel (between subunits A/B] and C/D) and perpendicular (between subunits A/C and B/D) interfaces according to the mutual orientations of the main axes of the β-barrels. Both interfaces involve hydrophobic and hydrophilic interactions (fig. 1).

[...]

Relatively at tertiary structure, the monomeric subunits within the tetramers are essentially identical. The overall backbone topology shows the typical 11-stranded b-barrel [sic] fold, with the central a-helix [sic] interrupted by the chromophore. The N-terminal end (residues 1–7) forms a lid on the same barrel, thereby assisting in shielding the interior of the can from the environment, whereas the C-terminal tail (residues 220–228) wraps around the other barrel in the A/B dimer. The AsGFP structure is similar to that of AvGFP. Backbone structural differences between AsGFP and AvGFP are most pronounced in the region corresponding to amino acids 138–141 (143–146 in AvGFP) and in the loop region formed by amino acids 195–206 (204–216 in AvGFP).

The chromophore of AsGFP is a planar resonance system formed autocatalytically by residues Gln63, Tyr64, and Gly65 (fig. 2, A and B). It consists of an imidazolinone ring generated by cyclization between the Gln63-C' and the Gly65-Nα atoms and the Tyr64 [7 hydroxyphenyl group, which is made coplanar with the imidazolinone due to dehydrogenation insaturation of its Cα-Cβ bond.]

The asymmetric unit of asFP499 contains two identical tetramers related by a noncrystallographic symmetry. They are arranged as dimers of dimers, so that two types of subunit interfaces can be distinguished; they are denoted as antiparallel (between subunits A/B and C/D) and perpendicular (between subunits A/C and B/D) interfaces according to the mutual orientations of the main axes of the β-barrels. Both interfaces involve hydrophobic and hydrophilic interactions.

[page 4212]

Tertiary structure

The monomeric subunits within the tetramers are essentially identical, as judged from the average root mean-square deviation (rmsd) of the Ca atoms of 0.21 A° . The overall backbone topology shows the typical 11-stranded β-barrel fold, with the central α-helix interrupted by the chromophore. The N-terminal end (residues 1–7) forms a lid on the same barrel, thereby assisting in shielding the interior of the can from the environment, whereas the C-terminal tail (residues 220–228) wraps around the other barrel in the A/B dimer. The asFP499 structure is similar to that of avGFP, with an rmsd of the Ca atoms of 1.15A° . Backbone structural differences between asFP499 and avGFP are most pronounced in the region corresponding to amino acids 138–141 (143–146 in avGFP) and in the loop region formed by amino acids 195–206 (204–216 in avGFP).

The chromophore and its environment

The chromophore of asFP499 is a planar resonance system formed autocatalytically by residues Gln63, Tyr64, and Gly65 (Fig. 1, A and B). It consists of an imidazolinone ring generated by cyclization between the Gln63-C9 and the Gly65-Nα atoms and the Tyr64 hydroxyphenyl group, which is made coplanar with the imidazolinone due to dehydrogenation insaturation of itsCα-Cβ bond.

Anmerkungen

The source is mentioned once in the beginning. However, it does not become clear that the entire page (except the figure 1) is copied and that the copy is almost literal.

Sichter
(SleepyHollow02), Hindemith

[2.] Tim/Fragment 007 01 - Diskussion
Zuletzt bearbeitet: 2014-10-25 05:55:06 Hindemith
BauernOpfer, Fragment, Gesichtet, Nienhaus 2006, SMWFragment, Schutzlevel sysop, Tim

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[It consists of an imidazolinone ring generated by cyclization between the Gln63-C' and the Gly65-Nα atoms and the Tyr64] hydroxyphenyl group, which is made coplanar with the imidazolinone due to dehydrogenation insaturation of its Cα-Cβ bond. Whereas there is a glutamine in the first position of the tripeptide instead of the serine in AvGFP, the AsGFP chromophore is essentially identical to that of AvGFP, including the cis configuration at the Tyr64-Cβ, which is encountered more frequently than the trans conformation.

Tim 007a diss.png

Fig. 2 (A) Top and (B) side view of the electron density map of the AsGFP chromophore and its environment, contoured at 1.2 s. (C) Close-up of the AsGFP chromophore (green, carbon; red, oxygen; blue, nitrogen) and surrounding residues (black, carbon; red, oxygen; blue, nitrogen). The backbone structures are plotted as lines; the side chains are accentuated. Water molecules are plotted as red spheres. Hydrogen bonds are represented by dashed lines. Distances are given in angstroms. (Nienhaus et al., 2006)

The chromophore of AsGFP is tightly encased within the β-barrel by a hydrogen bond network involving polar and charged residues and altogether 10 structural waters within a distance of 5 Angstrom from the imidazolinone oxygen.

Tim 007a source.png

Figure 1 (A) Top and (B) side view of the electron density map of the asFP499 chromophore and its environment, contoured at 1.2 s. (C) Close-up of the asFP499 chromophore (green, carbon; red, oxygen; blue, nitrogen) and surrounding residues (black, carbon; red, oxygen; blue, nitrogen). The backbone structures are plotted as lines; the side chains are accentuated. Water molecules are plotted as red spheres. Hydrogen bonds are represented by dashed lines. Distances are given in angstroms.

It consists of an imidazolinone ring generated by cyclization between the Gln63-C´ and the Gly65-Nα atoms and the Tyr64 hydroxyphenyl group, which is made coplanar with the imidazolinone due to dehydrogenation insaturation of its Cα-Cβ bond. Whereas there is a glutamine in the first position of the tripeptide instead of the serine in avGFP, the asFP499 chromophore is essentially identical to that of avGFP, including the cis configuration at the Tyr64-Cβ, which is encountered more frequently than the trans conformation. The chromophore of asFP499 is tightly encased within the β-barrel by a hydrogen-bond network involving polar and charged residues and altogether 10 structural waters within a

[page 4213]

distance of 5 A° from the imidazolinone oxygen.

Anmerkungen

The source is given for the figure and its caption, but not for the remainder of the text.

Sichter
(SleepyHollow02), Hindemith

[3.] Tim/Fragment 008 01 - Diskussion
Zuletzt bearbeitet: 2014-10-25 05:59:20 Hindemith
BauernOpfer, Fragment, Gesichtet, Nienhaus 2006, SMWFragment, Schutzlevel sysop, Tim

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Figure 2 C displays the chromophore cage, with potential hydrogen-bond interactions represented by dashed lines. The highly conserved residues Arg92 and Glu212 have been implicated as being crucially involved in the mechanism of autocatalytic chromophore formation (Ormo et al., 1996; Sniegowski et al., 2005; Wood et al., 2005). The Arg92 guanidinium group hydrogen bonds to the Tyr64-derived carbonyl oxygen, whereas Glu212 is positioned within hydrogen-bonding distance to the heterocyclic ring nitrogen. Fig. 1 C displays the chromophore cage, with potential hydrogen-bond interactions represented by dashed lines. [...] The highly conserved residues Arg92 and Glu212 have been implicated as being crucially involved in the mechanism of autocatalytic chromophore formation (6,40,41). The Arg92 guanidinium group hydrogen bonds to the Tyr64-derived carbonyl oxygen, whereas Glu212 is positioned within hydrogen-bonding distance to the heterocyclic ring nitrogen, as has been observed earlier for yellow avGFP variants (42)

6. Ormo¨ , M., A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington. 1996. Crystal structure of the Aequorea victoria green fluorescent protein. Science. 273:1392–1395.

40. Sniegowski, J. A., J. W. Lappe, H. N. Patel, H. A. Huffman, and R. M. Wachter. 2005. Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein. J. Biol. Chem. 280: 26248–26255.

41. Wood, T. I., D. P. Barondeau, C. Hitomi, C. J. Kassmann, J. A. Tainer, and E. D. Getzoff. 2005. Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis. Biochemistry. 44:16211– 16220.

42. Wachter, R. M., M. A. Elsliger, K. Kallio, G. T. Hanson, and S. J. Remington. 1998. Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. Structure. 6: 1267–1277.

Anmerkungen

The source is given the previous page to reference the figure that is described here. Nothing indicates, however, that this section is also taken from the source.

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(SleepyHollow02), Hindemith

[4.] Tim/Fragment 012 01 - Diskussion
Zuletzt bearbeitet: 2014-10-25 06:42:18 Hindemith
BauernOpfer, Fragment, Gesichtet, Nienhaus 2006, SMWFragment, Schutzlevel sysop, Tim

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[At pH ˃ 10, a red-]shifted [sic] emerged, with absorbance and emission peaks displaced by 18 and 10 nm to the red, respectively. At pH ˂ 4 and pH ˃ 12 the protein was thermodynamically unstable.

[...]

In analogy to wild-type AvGFP, the AsGFP499 protein showed two bands in the UV/visible spectrum over a wide pH range. Although initially debated (Voityuk et al., 1998), there is now general agreement that the A and B bands are associated with the neutral and anionic states of chromophore (Tsien, 1998). In AsGFP499, the two bands were similar in area in the entire pH range in which the protein was stable; and remarkably, the protonated form of the chromophore became more dominant with increasing pH.

In the structure of AsGFP499, amino acid Glu212 is shown to be hydrogen bonded to the chromophore heterocycle and not connected to the phenolic oxygen at all. Therefore, proton transfer between the chromophore and Glu212 cannot take place. In addition to hydrogen bonds to two water molecules, the Tyr64 phenol oxygen is connected to the Ser143 hydroxyl via a short hydrogen bond, which in turn is hydrogen bonded to Asp158.

At pH . 10, a red-shifted form emerges, with absorbance and emission peaks displaced by 18 and 10 nm to the red, respectively (Fig. 2 D).

[page 4215]

In analogy to wild-type avGFP, the asFP499 protein shows two bands in the UV/visible spectrum over a wide pH range (Fig. 4). Although initially debated (51,52), there is now general agreement that the A and B bands are associated with the neutral and anionic states of chromophore (9). [...]

[...]

In asFP499, the two bands are similar in area in the entire pH range in which the protein is stable (Fig. 4); and remarkably, the protonated form of the chromophore becomes more dominant with increasing pH. In the structure of asFP499, amino acid Glu212 (corresponding to Glu222 in avGFP) is shown to be hydrogen bonded to the chromophore heterocycle and not connected to the phenolic oxygen at all. Therefore, proton transfer between the chromophore and Glu212 cannot take place. However, the structure in Fig. 1 C suggests an alternative explanation for the appearance of the two conformations. In addition to hydrogen bonds to two water molecules, the Tyr64 phenol oxygen is connected to the

[page 4216]

Ser143 hydroxyl via a short hydrogen bond (2.5 A° ), which in turn is hydrogen bonded to Asp158 (2.7 A° ).


9. Tsien, R. Y. 1998. The green fluorescent protein. Annu. Rev. Biochem. 67:509–544.

51. Weber, W., V. Helms, J. A. McCammon, and P. W. Langhoff. 1999. Shedding light on the dark and weakly fluorescent states of green fluorescent proteins. Proc. Natl. Acad. Sci. USA. 96:6177–6182.

52. Voityuk, A. A., M. E. Michel-Beyerle, and N. Ro¨sch. 1998. Quantum chemical modeling of structure and absorption spectra of the chromophore in green fluorescent proteins. Chem. Phys. 231:13–25.

Anmerkungen

The source is given on the previous page. The reader may assume that the work of Nienhaus et al. (2006) is described in the first two paragraphs, but not that this is done in the words of Nienhaus et al. (2006). The last documented paragraph however is not written in the descriptive past tense but rather makes factual statements, such that the reader has no way of knowing that he is reading the words of Nienhaus et al. (2006).

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(SleepyHollow02), Hindemith

[5.] Tim/Fragment 013 01 - Diskussion
Zuletzt bearbeitet: 2014-10-25 07:16:35 Hindemith
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The schemes in figure 5, A and B, show the tightly coupled system of two protonatable groups between which protons can be shuttled. The small ratio between neutral and anionic population implies that only slight differences in free energies exist between the two conformations in the electronic ground state.

Tim 013a diss.png

Fig. 5. Schematic representation of the different protonation states of the AsGFP499 chromophore and its environment that are proposed to cause the spectral changes. (Nienhaus et al., 2006).

Upon photon absorption, this balance is disturbed. Phenols typically become more acidic upon electronic excitation (Tsien, 1998; Voityuk et al., 1998); and therefore, we expect efficient excited state proton transfer (ESPT) to Asp158, as is inferred from the observation that excitation in the A and B bands is equally efficient for fluorescence in the 499nm emission band for pH ˂ 8. Evidently, protonation of Asp158 is a key ingredient in the proton shuttling mechanism described above.

The schemes in Fig. 6, A and B, show the tightly coupled system of two protonatable groups between which protons can be shuttled. The small ratio between neutral and anionic population implies that only slight differences in free energies exist between the two conformations in the electronic ground state. Upon photon absorption, this balance is disturbed. Phenols typically become more acidic upon electronic excitation (9,52); and therefore, we expect efficient excited state proton transfer (ESPT) to Asp158, as is inferred from the observation that excitation in the A and B bands is equally efficient for fluorescence in the 499-nm emission band for pH < 8.

To further support the model presented in Fig. 6 by experimental evidence, we have produced the mutant Asp158Asn, which has its protonatable carboxyl residue replaced by a nonprotonatable carboxamide. Evidently, protonation of Asp158 is a key ingredient in the proton shuttling mechanism described above.

Tim 013a source.png

FIGURE 6 Schematic representation of the different protonation states of the asFP499 chromophore and its environment that are proposed to cause the spectral changes in Fig. 4.


9. Tsien, R. Y. 1998. The green fluorescent protein. Annu. Rev. Biochem. 67:509–544.

52. Voityuk, A. A., M. E. Michel-Beyerle, and N. Ro¨sch. 1998. Quantum chemical modeling of structure and absorption spectra of the chromophore in green fluorescent proteins. Chem. Phys. 231:13–25.

Anmerkungen

The source is given for the figure and its caption, but not for the remainder of the text.

Sichter
(SleepyHollow02), Hindemith

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