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The effect of amino acid deletions and substitutions in the longest loop of GFP.

Flores-Ramírez G, Rivera M, Morales-Pablos A, Osuna J, Soberón X, Gaytán P - BMC Chem Biol (2007)

Bottom Line: The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties.In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated.Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ap, Postal 510-3 Cuernavaca, Morelos 62250, México. gabyflo32@hotmail.com

ABSTRACT

Background: The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region.

Results: In this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Delta I129 and sgGFP-Delta D130. Interestingly, both mutants were thermosensitive and at 30 degrees C sgGFP-Delta D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein.

Conclusion: The behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure.

No MeSH data available.


"Temperature denaturation curves of the purified proteins sgGFP wt, sgGFP-ΔI129 and sgGFP-ΔD130. Triplicated aliquots of each sample were subjected to different temperatures (25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C) during 5 minutes and the initial (Fo) and final fluorescence (Ff) was measured. The ratio Ff/Fo versus temperature was plotted."
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Figure 4: "Temperature denaturation curves of the purified proteins sgGFP wt, sgGFP-ΔI129 and sgGFP-ΔD130. Triplicated aliquots of each sample were subjected to different temperatures (25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C) during 5 minutes and the initial (Fo) and final fluorescence (Ff) was measured. The ratio Ff/Fo versus temperature was plotted."

Mentions: Temperature denaturation curves (see Figure 4) for the active purified mutants sgGFP-Δ I129 and sgGFP-Δ D130, demonstrated that these proteins are less heat stable than the parent protein, but not enough to give account for the significative protein reduction at 37°C. Therefore, these amino acids are essential for good folding, especially D130.


The effect of amino acid deletions and substitutions in the longest loop of GFP.

Flores-Ramírez G, Rivera M, Morales-Pablos A, Osuna J, Soberón X, Gaytán P - BMC Chem Biol (2007)

"Temperature denaturation curves of the purified proteins sgGFP wt, sgGFP-ΔI129 and sgGFP-ΔD130. Triplicated aliquots of each sample were subjected to different temperatures (25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C) during 5 minutes and the initial (Fo) and final fluorescence (Ff) was measured. The ratio Ff/Fo versus temperature was plotted."
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919350&req=5

Figure 4: "Temperature denaturation curves of the purified proteins sgGFP wt, sgGFP-ΔI129 and sgGFP-ΔD130. Triplicated aliquots of each sample were subjected to different temperatures (25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C) during 5 minutes and the initial (Fo) and final fluorescence (Ff) was measured. The ratio Ff/Fo versus temperature was plotted."
Mentions: Temperature denaturation curves (see Figure 4) for the active purified mutants sgGFP-Δ I129 and sgGFP-Δ D130, demonstrated that these proteins are less heat stable than the parent protein, but not enough to give account for the significative protein reduction at 37°C. Therefore, these amino acids are essential for good folding, especially D130.

Bottom Line: The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties.In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated.Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ap, Postal 510-3 Cuernavaca, Morelos 62250, México. gabyflo32@hotmail.com

ABSTRACT

Background: The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region.

Results: In this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Delta I129 and sgGFP-Delta D130. Interestingly, both mutants were thermosensitive and at 30 degrees C sgGFP-Delta D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein.

Conclusion: The behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure.

No MeSH data available.