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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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Schematic outline of the chromophores and selected neighboring residues in GFP (A), DsRed (B, D), and DsRed-NF (C, E, F) in "sticks" and "spacefill" representation. Carbon atoms are gray, nitrogen atoms are blue, and oxygen atoms are red. Images were generated by RasMol 2.6 software. Computer modeling for DsRed-NF was performed using Swiss-PdbViewer and HyperChem 5.01 software.
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Figure 2: Schematic outline of the chromophores and selected neighboring residues in GFP (A), DsRed (B, D), and DsRed-NF (C, E, F) in "sticks" and "spacefill" representation. Carbon atoms are gray, nitrogen atoms are blue, and oxygen atoms are red. Images were generated by RasMol 2.6 software. Computer modeling for DsRed-NF was performed using Swiss-PdbViewer and HyperChem 5.01 software.

Mentions: Although sequence comparison of known GFP-like proteins does not reveal absolutely invariable differences between FPs and CPs, one can draw attention to three positions, specifically, 148, 165, and 203, which are occupied by noticeably different residues in the two types of proteins (Fig. 1, Table 1). Since residues at these positions are in a close proximity to chromophore (Fig. 2A,2B) [7-10], it is reasonable to presume that they can participate in the determination of the state (fluorescent or non-fluorescent) of a particular protein.


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

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

Schematic outline of the chromophores and selected neighboring residues in GFP (A), DsRed (B, D), and DsRed-NF (C, E, F) in "sticks" and "spacefill" representation. Carbon atoms are gray, nitrogen atoms are blue, and oxygen atoms are red. Images were generated by RasMol 2.6 software. Computer modeling for DsRed-NF was performed using Swiss-PdbViewer and HyperChem 5.01 software.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Schematic outline of the chromophores and selected neighboring residues in GFP (A), DsRed (B, D), and DsRed-NF (C, E, F) in "sticks" and "spacefill" representation. Carbon atoms are gray, nitrogen atoms are blue, and oxygen atoms are red. Images were generated by RasMol 2.6 software. Computer modeling for DsRed-NF was performed using Swiss-PdbViewer and HyperChem 5.01 software.
Mentions: Although sequence comparison of known GFP-like proteins does not reveal absolutely invariable differences between FPs and CPs, one can draw attention to three positions, specifically, 148, 165, and 203, which are occupied by noticeably different residues in the two types of proteins (Fig. 1, Table 1). Since residues at these positions are in a close proximity to chromophore (Fig. 2A,2B) [7-10], it is reasonable to presume that they can participate in the determination of the state (fluorescent or non-fluorescent) of a particular protein.

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