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GFP Loss-of-Function Mutations in Arabidopsis thaliana.

Fu JL, Kanno T, Liang SC, Matzke AJ, Matzke M - G3 (Bethesda) (2015)

Bottom Line: Mutations that alter residues important for chromophore maturation, such as Arg96 and Ser205, greatly reduce or extinguish fluorescence without dramatically altering GFP protein accumulation.Many mutations in this category generate substitutions of highly conserved glycine residues, including the following: Gly67 in the chromogenic tripeptide; Gly31, Gly33, and Gly35 in the second β-strand; and Gly20, Gly91, and Gly127 in the lids of the β-barrel scaffold.Our genetic analysis supports conclusions from structural and biochemical studies and demonstrates a critical role for multiple, highly conserved glycine residues in GFP protein stability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant and Microbial Biology, Academia Sinica 128, Section 2, Academia Road, Nangang District, Taipei 115, Taiwan.

No MeSH data available.


Related in: MedlinePlus

Overall GFP fold and positions of amino acid substitutions leading to loss of fluorescence. A schematic depiction of the overall fold of Thr65-GFP protein (Yang et al. 1996) is redrawn here. The vertical arrows indicate the 11 β strands of the β-barrel structure. Amino acid residue numbers at the base and tips of the arrows indicate the beginning and ends of secondary structural elements. The chromogenic tripeptide (Thr65-Tyr66-Gly67) is positioned on an internal α-helix (diagonal cylinder) extending from amino acids 56 to 72. Amino acid substitutions identified in our screen that lead to losses of fluorescence are indicated. Solid red outlines denote substitutions causing defects in chromophore formation without substantial reductions in GFP protein accumulation. Dotted red outlines indicate substitutions resulting in lowered levels of GFP protein accumulation relative to wild-type. For the remaining substitutions, no GFP protein was detected by Western blotting under the conditions used. Lid residues at the N and C termini (G91 and G127) and the opposite side (G20), which is referred to as the "top" of the barrel (Zimmer et al. 2014), are highlighted in blue.
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fig1: Overall GFP fold and positions of amino acid substitutions leading to loss of fluorescence. A schematic depiction of the overall fold of Thr65-GFP protein (Yang et al. 1996) is redrawn here. The vertical arrows indicate the 11 β strands of the β-barrel structure. Amino acid residue numbers at the base and tips of the arrows indicate the beginning and ends of secondary structural elements. The chromogenic tripeptide (Thr65-Tyr66-Gly67) is positioned on an internal α-helix (diagonal cylinder) extending from amino acids 56 to 72. Amino acid substitutions identified in our screen that lead to losses of fluorescence are indicated. Solid red outlines denote substitutions causing defects in chromophore formation without substantial reductions in GFP protein accumulation. Dotted red outlines indicate substitutions resulting in lowered levels of GFP protein accumulation relative to wild-type. For the remaining substitutions, no GFP protein was detected by Western blotting under the conditions used. Lid residues at the N and C termini (G91 and G127) and the opposite side (G20), which is referred to as the "top" of the barrel (Zimmer et al. 2014), are highlighted in blue.

Mentions: From the chromogenic tripeptide (Thr65-Tyr66-Gly67), we recovered substitutions of Thr65 (T65I) and Gly67 (G67S and G67D) (Table 1, Figure 1). Because the only EMS-induced mutation of tyrosine codons produces a silent mutation (Table S1), it was not possible to obtain substitutions of highly conserved Tyr66 in our screen. Gly67 is also among the 23 highly conserved amino acids in GFP-related proteins (Ong et al. 2011). Gly67 mutant plants did not show visible GFP fluorescence (Table 1, Figure S2), although they did accumulate detectable, albeit reduced, levels of GFP protein (Figure 2A), suggesting that the G67 substitutions compromise to some extent GFP protein stability. The lack of visible fluorescence despite detectable protein accumulation is consistent with previous results showing that substituting Gly67 with any other amino acid prevents chromophore formation (Lemay et al. 2008; Stepanenko et al. 2013). Glycine is unique in having H as a side chain, in contrast to other amino acids that have a carbon, endowing glycine with exceptional conformational flexibility (Betts and Russell, 2003). Thus, glycine is the only amino acid at position 67 that permits formation of a kinked internal α-helix, which places Gly67 close to the residue at position 65 for nucleophilic attack during chromophore synthesis (Lemay et al. 2008; Stepanenko et al. 2013).


GFP Loss-of-Function Mutations in Arabidopsis thaliana.

Fu JL, Kanno T, Liang SC, Matzke AJ, Matzke M - G3 (Bethesda) (2015)

Overall GFP fold and positions of amino acid substitutions leading to loss of fluorescence. A schematic depiction of the overall fold of Thr65-GFP protein (Yang et al. 1996) is redrawn here. The vertical arrows indicate the 11 β strands of the β-barrel structure. Amino acid residue numbers at the base and tips of the arrows indicate the beginning and ends of secondary structural elements. The chromogenic tripeptide (Thr65-Tyr66-Gly67) is positioned on an internal α-helix (diagonal cylinder) extending from amino acids 56 to 72. Amino acid substitutions identified in our screen that lead to losses of fluorescence are indicated. Solid red outlines denote substitutions causing defects in chromophore formation without substantial reductions in GFP protein accumulation. Dotted red outlines indicate substitutions resulting in lowered levels of GFP protein accumulation relative to wild-type. For the remaining substitutions, no GFP protein was detected by Western blotting under the conditions used. Lid residues at the N and C termini (G91 and G127) and the opposite side (G20), which is referred to as the "top" of the barrel (Zimmer et al. 2014), are highlighted in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555221&req=5

fig1: Overall GFP fold and positions of amino acid substitutions leading to loss of fluorescence. A schematic depiction of the overall fold of Thr65-GFP protein (Yang et al. 1996) is redrawn here. The vertical arrows indicate the 11 β strands of the β-barrel structure. Amino acid residue numbers at the base and tips of the arrows indicate the beginning and ends of secondary structural elements. The chromogenic tripeptide (Thr65-Tyr66-Gly67) is positioned on an internal α-helix (diagonal cylinder) extending from amino acids 56 to 72. Amino acid substitutions identified in our screen that lead to losses of fluorescence are indicated. Solid red outlines denote substitutions causing defects in chromophore formation without substantial reductions in GFP protein accumulation. Dotted red outlines indicate substitutions resulting in lowered levels of GFP protein accumulation relative to wild-type. For the remaining substitutions, no GFP protein was detected by Western blotting under the conditions used. Lid residues at the N and C termini (G91 and G127) and the opposite side (G20), which is referred to as the "top" of the barrel (Zimmer et al. 2014), are highlighted in blue.
Mentions: From the chromogenic tripeptide (Thr65-Tyr66-Gly67), we recovered substitutions of Thr65 (T65I) and Gly67 (G67S and G67D) (Table 1, Figure 1). Because the only EMS-induced mutation of tyrosine codons produces a silent mutation (Table S1), it was not possible to obtain substitutions of highly conserved Tyr66 in our screen. Gly67 is also among the 23 highly conserved amino acids in GFP-related proteins (Ong et al. 2011). Gly67 mutant plants did not show visible GFP fluorescence (Table 1, Figure S2), although they did accumulate detectable, albeit reduced, levels of GFP protein (Figure 2A), suggesting that the G67 substitutions compromise to some extent GFP protein stability. The lack of visible fluorescence despite detectable protein accumulation is consistent with previous results showing that substituting Gly67 with any other amino acid prevents chromophore formation (Lemay et al. 2008; Stepanenko et al. 2013). Glycine is unique in having H as a side chain, in contrast to other amino acids that have a carbon, endowing glycine with exceptional conformational flexibility (Betts and Russell, 2003). Thus, glycine is the only amino acid at position 67 that permits formation of a kinked internal α-helix, which places Gly67 close to the residue at position 65 for nucleophilic attack during chromophore synthesis (Lemay et al. 2008; Stepanenko et al. 2013).

Bottom Line: Mutations that alter residues important for chromophore maturation, such as Arg96 and Ser205, greatly reduce or extinguish fluorescence without dramatically altering GFP protein accumulation.Many mutations in this category generate substitutions of highly conserved glycine residues, including the following: Gly67 in the chromogenic tripeptide; Gly31, Gly33, and Gly35 in the second β-strand; and Gly20, Gly91, and Gly127 in the lids of the β-barrel scaffold.Our genetic analysis supports conclusions from structural and biochemical studies and demonstrates a critical role for multiple, highly conserved glycine residues in GFP protein stability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant and Microbial Biology, Academia Sinica 128, Section 2, Academia Road, Nangang District, Taipei 115, Taiwan.

No MeSH data available.


Related in: MedlinePlus