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Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis.

Bulina ME, Chudakov DM, Mudrik NN, Lukyanov KA - BMC Biochem. (2002)

Bottom Line: For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence.For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs.We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117997 Moscow, Russia. biomasha@mail.ru

ABSTRACT

Background: Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date.

Results: Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein.

Conclusions: We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.

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Normalized spectra for selected mutants of asCP and DsRed. Absorption (black solid lines), excitation (colored dashed lines), and emission (colored solid lines) spectra are shown for each mutant. Blue, green, or red excitation-emission lines correspond to color of fluorescence. (A) asCP-S165V. Blue fluorescence is about tenfold weaker than red. (B) asCP-S165A. (C) asCP-S165C. (D) asCP-S165T. Green emision is about twofold stronger than red. (E) DsRed-NF. Green emission peak is about threefold lower than red one.
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Figure 3: Normalized spectra for selected mutants of asCP and DsRed. Absorption (black solid lines), excitation (colored dashed lines), and emission (colored solid lines) spectra are shown for each mutant. Blue, green, or red excitation-emission lines correspond to color of fluorescence. (A) asCP-S165V. Blue fluorescence is about tenfold weaker than red. (B) asCP-S165A. (C) asCP-S165C. (D) asCP-S165T. Green emision is about twofold stronger than red. (E) DsRed-NF. Green emission peak is about threefold lower than red one.

Mentions: First of all, we tested a substitution S165V because several FPs carry Val at this position. This mutation resulted in the appearance of a clearly visible red fluorescence with a maximum at 620 nm (Fig. 3A, Table 2). Interestingly, in comparison with the wild type asCP, the mutant asCP-S165V showed a strongly modified absorption spectrum which included an additional peak at 390 nm. Absorption at this wavelength produced a very weak (about 10-fold less than the red fluorescence) blue fluorescence at 465 nm.


Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis.

Bulina ME, Chudakov DM, Mudrik NN, Lukyanov KA - BMC Biochem. (2002)

Normalized spectra for selected mutants of asCP and DsRed. Absorption (black solid lines), excitation (colored dashed lines), and emission (colored solid lines) spectra are shown for each mutant. Blue, green, or red excitation-emission lines correspond to color of fluorescence. (A) asCP-S165V. Blue fluorescence is about tenfold weaker than red. (B) asCP-S165A. (C) asCP-S165C. (D) asCP-S165T. Green emision is about twofold stronger than red. (E) DsRed-NF. Green emission peak is about threefold lower than red one.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC113743&req=5

Figure 3: Normalized spectra for selected mutants of asCP and DsRed. Absorption (black solid lines), excitation (colored dashed lines), and emission (colored solid lines) spectra are shown for each mutant. Blue, green, or red excitation-emission lines correspond to color of fluorescence. (A) asCP-S165V. Blue fluorescence is about tenfold weaker than red. (B) asCP-S165A. (C) asCP-S165C. (D) asCP-S165T. Green emision is about twofold stronger than red. (E) DsRed-NF. Green emission peak is about threefold lower than red one.
Mentions: First of all, we tested a substitution S165V because several FPs carry Val at this position. This mutation resulted in the appearance of a clearly visible red fluorescence with a maximum at 620 nm (Fig. 3A, Table 2). Interestingly, in comparison with the wild type asCP, the mutant asCP-S165V showed a strongly modified absorption spectrum which included an additional peak at 390 nm. Absorption at this wavelength produced a very weak (about 10-fold less than the red fluorescence) blue fluorescence at 465 nm.

Bottom Line: For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence.For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs.We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117997 Moscow, Russia. biomasha@mail.ru

ABSTRACT

Background: Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date.

Results: Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein.

Conclusions: We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.

Show MeSH
Related in: MedlinePlus