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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.


Western blotting analysis of deleted mutants grown at two different temperatures. (A) Single amino acid deletions comprised in the region 129–142 of sgGFP and the double mutant sgGFP-ΔI129/ΔD130 grown at 37°C. (B) Some sgGFP mutants grown at 30°C. S and P represent the soluble and insoluble fraction of the cells, respectively. Fractions S and P were run on different gels. Lane 1: sgGFP wt, lane 2: sgGFP-ΔI129, lane 3: sgGFP-ΔD130, lane 4: sgGFP-ΔF131, lane 5: sgGFP-ΔK132, lane 6: sgGFP-ΔE133, lane 7: sgGFP-ΔD134, lane 8: sgGFP-ΔG135, lane 9: sgGFP-ΔN136, lane 10: sgGFP-ΔI137, lane 11: sgGFP-ΔL138, lane 12: sgGFP-ΔG139, lane 13: sgGFP-ΔH140, lane 14: sgGFP-ΔK141, lane 15: sgGFP-ΔL142 and lane 16: sgGFP-ΔI129/ΔD130.
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Figure 3: Western blotting analysis of deleted mutants grown at two different temperatures. (A) Single amino acid deletions comprised in the region 129–142 of sgGFP and the double mutant sgGFP-ΔI129/ΔD130 grown at 37°C. (B) Some sgGFP mutants grown at 30°C. S and P represent the soluble and insoluble fraction of the cells, respectively. Fractions S and P were run on different gels. Lane 1: sgGFP wt, lane 2: sgGFP-ΔI129, lane 3: sgGFP-ΔD130, lane 4: sgGFP-ΔF131, lane 5: sgGFP-ΔK132, lane 6: sgGFP-ΔE133, lane 7: sgGFP-ΔD134, lane 8: sgGFP-ΔG135, lane 9: sgGFP-ΔN136, lane 10: sgGFP-ΔI137, lane 11: sgGFP-ΔL138, lane 12: sgGFP-ΔG139, lane 13: sgGFP-ΔH140, lane 14: sgGFP-ΔK141, lane 15: sgGFP-ΔL142 and lane 16: sgGFP-ΔI129/ΔD130.

Mentions: Additional characterization of whole cells containing the mutants sgGFP-Δ I129 and sgGFP-Δ D130 revealed that both proteins suffered a blue-shift of two nanometers in their maximum emission and their fluorescence intensity was reduced to 21% and 17%, respectively, relative to wt sgGFP. The last result did not correlate with the phenotype observed in plates, where the green color of the mutants was only slightly less intense than the wild-type protein. We then decided to measure the quantum yield of the mutant proteins, which turned out to be 31% and 21% smaller than the parent protein, respectively. Because the quantum yield decrement of the mutants did not fully account for the fluorescence loss, we turned our attention towards protein concentration in the cells, another factor that affects fluorescence intensity. The amount of soluble and non-soluble protein for each mutant was analyzed by western blotting as shown in Figure 3, using anti-GFP for the detection. This experiment clearly revealed that the main reason for the reduction or loss of fluorescence of the mutants was their low concentration which, in turn, could also be due to low stability or incorrect folding [25]. Not surprisingly, sgGFP-Δ I129 and sgGFP-Δ D130 were the best mutants expressed. To assess if the proteins were inactivated by improper folding we grew the mutants at 30°C. At this lower temperature, the fluorescence of sgGFP-Δ I129 increased from 21% to 46%, whereas sgGFP-Δ D130 increased from 17% to 116% as compared to wt sgGFP. These results indicated that both deletion mutants are thermosensitive, and even more, at lower temperatures sgGFP-Δ D130 is more fluorescent than the wild-type protein. Lower temperatures frequently favor appropriate folding of mutants [13]. Western blotting of the mutants grown at 30°C, shown on Figure 3b, confirmed that the protein concentration was increased.


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)

Western blotting analysis of deleted mutants grown at two different temperatures. (A) Single amino acid deletions comprised in the region 129–142 of sgGFP and the double mutant sgGFP-ΔI129/ΔD130 grown at 37°C. (B) Some sgGFP mutants grown at 30°C. S and P represent the soluble and insoluble fraction of the cells, respectively. Fractions S and P were run on different gels. Lane 1: sgGFP wt, lane 2: sgGFP-ΔI129, lane 3: sgGFP-ΔD130, lane 4: sgGFP-ΔF131, lane 5: sgGFP-ΔK132, lane 6: sgGFP-ΔE133, lane 7: sgGFP-ΔD134, lane 8: sgGFP-ΔG135, lane 9: sgGFP-ΔN136, lane 10: sgGFP-ΔI137, lane 11: sgGFP-ΔL138, lane 12: sgGFP-ΔG139, lane 13: sgGFP-ΔH140, lane 14: sgGFP-ΔK141, lane 15: sgGFP-ΔL142 and lane 16: sgGFP-ΔI129/ΔD130.
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Related In: Results  -  Collection

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Figure 3: Western blotting analysis of deleted mutants grown at two different temperatures. (A) Single amino acid deletions comprised in the region 129–142 of sgGFP and the double mutant sgGFP-ΔI129/ΔD130 grown at 37°C. (B) Some sgGFP mutants grown at 30°C. S and P represent the soluble and insoluble fraction of the cells, respectively. Fractions S and P were run on different gels. Lane 1: sgGFP wt, lane 2: sgGFP-ΔI129, lane 3: sgGFP-ΔD130, lane 4: sgGFP-ΔF131, lane 5: sgGFP-ΔK132, lane 6: sgGFP-ΔE133, lane 7: sgGFP-ΔD134, lane 8: sgGFP-ΔG135, lane 9: sgGFP-ΔN136, lane 10: sgGFP-ΔI137, lane 11: sgGFP-ΔL138, lane 12: sgGFP-ΔG139, lane 13: sgGFP-ΔH140, lane 14: sgGFP-ΔK141, lane 15: sgGFP-ΔL142 and lane 16: sgGFP-ΔI129/ΔD130.
Mentions: Additional characterization of whole cells containing the mutants sgGFP-Δ I129 and sgGFP-Δ D130 revealed that both proteins suffered a blue-shift of two nanometers in their maximum emission and their fluorescence intensity was reduced to 21% and 17%, respectively, relative to wt sgGFP. The last result did not correlate with the phenotype observed in plates, where the green color of the mutants was only slightly less intense than the wild-type protein. We then decided to measure the quantum yield of the mutant proteins, which turned out to be 31% and 21% smaller than the parent protein, respectively. Because the quantum yield decrement of the mutants did not fully account for the fluorescence loss, we turned our attention towards protein concentration in the cells, another factor that affects fluorescence intensity. The amount of soluble and non-soluble protein for each mutant was analyzed by western blotting as shown in Figure 3, using anti-GFP for the detection. This experiment clearly revealed that the main reason for the reduction or loss of fluorescence of the mutants was their low concentration which, in turn, could also be due to low stability or incorrect folding [25]. Not surprisingly, sgGFP-Δ I129 and sgGFP-Δ D130 were the best mutants expressed. To assess if the proteins were inactivated by improper folding we grew the mutants at 30°C. At this lower temperature, the fluorescence of sgGFP-Δ I129 increased from 21% to 46%, whereas sgGFP-Δ D130 increased from 17% to 116% as compared to wt sgGFP. These results indicated that both deletion mutants are thermosensitive, and even more, at lower temperatures sgGFP-Δ D130 is more fluorescent than the wild-type protein. Lower temperatures frequently favor appropriate folding of mutants [13]. Western blotting of the mutants grown at 30°C, shown on Figure 3b, confirmed that the protein concentration was increased.

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.