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Structural plasticity of green fluorescent protein to amino acid deletions and fluorescence rescue by folding-enhancing mutations.

Liu SS, Wei X, Dong X, Xu L, Liu J, Jiang B - BMC Biochem. (2015)

Bottom Line: Other approach to reducing structural constraints may include minimizing the structure of GFPs.In this study, we interrogated the structural plasticity of a UV-optimized GFP variant (GFP(UV)) to amino acid deletions, characterized the effects of deletions and explored the feasibility of rescuing the fluorescence of deletion mutants using folding-enhancing mutations.Our results suggested that a "size-minimized" GFP may be developed by iterative incorporation of amino acid deletions, followed by fluorescence rescue with folding-enhancing mutations.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China. liushs@shanghaitech.edu.cn.

ABSTRACT

Background: Green fluorescent protein (GFP) and its derivative fluorescent proteins (FPs) are among the most commonly used reporter systems for studying gene expression and protein interaction in biomedical research. Most commercially available FPs have been optimized for their oligomerization state to prevent potential structural constraints that may interfere with the native function of fused proteins. Other approach to reducing structural constraints may include minimizing the structure of GFPs. Previous studies in an enhanced GFP variant (EGFP) identified a series of deletions that can retain GFP fluorescence. In this study, we interrogated the structural plasticity of a UV-optimized GFP variant (GFP(UV)) to amino acid deletions, characterized the effects of deletions and explored the feasibility of rescuing the fluorescence of deletion mutants using folding-enhancing mutations.

Methods: Transposon mutagenesis was used to screen amino acid deletions in GFP that led to fluorescent and nonfluorescent phenotypes. The fluorescent GFP mutants were characterized for their whole-cell fluorescence and fraction soluble. Fluorescent GFP mutants with internal deletions were purified and characterized for their spectral and folding properties. Folding-ehancing mutations were introduced to deletion mutants to rescue their compromised fluorescence.

Results: We identified twelve amino acid deletions that can retain the fluorescence of GFP(UV). Seven of these deletions are either at the N- or C- terminus, while the other five are located at internal helices or strands. Further analysis suggested that the five internal deletions diminished the efficiency of protein folding and chromophore maturation. Protein expression under hypothermic condition or incorporation of folding-enhancing mutations could rescue the compromised fluorescence of deletion mutants. In addition, we generated dual deletion mutants that can retain GFP fluorescence.

Conclusion: Our results suggested that a "size-minimized" GFP may be developed by iterative incorporation of amino acid deletions, followed by fluorescence rescue with folding-enhancing mutations.

No MeSH data available.


Related in: MedlinePlus

Excitation and emission spectra of purified GPFUV variants. a Purified wtGFPUV and deletion mutants, resolved on 12 % SDS-PAGE. M, marker. Lane 1 to 6, wtGFPUV, C48Δ, P75Δ, P75/D76➔H, E172Δ and S175/V176➔F, respectively. b Normalized excitation spectra. c Normalized emission spectra. Note that the spectra of these variants are largely overlapped
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Fig4: Excitation and emission spectra of purified GPFUV variants. a Purified wtGFPUV and deletion mutants, resolved on 12 % SDS-PAGE. M, marker. Lane 1 to 6, wtGFPUV, C48Δ, P75Δ, P75/D76➔H, E172Δ and S175/V176➔F, respectively. b Normalized excitation spectra. c Normalized emission spectra. Note that the spectra of these variants are largely overlapped

Mentions: Deletion mutants C48Δ, P75/D76➔H, P75Δ, E172Δ, S175/V176➔F were purified to more than 95 % homogeneity (Fig. 4a). The emission spectra of all variants under 397 and 475 nm excitation can be superimposed with that of wild-type GFPUV (wtGFPUV) (Fig. 4b), suggesting that the chromophores of these mutants did not have structural alteration. For excitation spectra, two peaks (397 and 475 nm) were observed for all samples. These deletion mutants showed variations in the intensity ratio between the major and minor peaks. For example, P75Δ showed slightly increased minor peak whereas the minor peak in S175/V176➔F mutant was considerably reduced (Fig. 4c). The 395 nm and 475 nm excitation peaks represent neutral and anionic chromophore, respectively [20]. Mutations that alter the ratio of the two chromophore species are frequently found in GFP variants. In one case, enhanced GFP (EGFP) only has a single excitation peak at 475 nm [21] because S65T mutation transform the chromophore to be completely ionized [20]. It is interesting to observe in this study that amino acid deletions, in addition to substitutions, can have impact on the ionization state of chromophore.Fig. 4


Structural plasticity of green fluorescent protein to amino acid deletions and fluorescence rescue by folding-enhancing mutations.

Liu SS, Wei X, Dong X, Xu L, Liu J, Jiang B - BMC Biochem. (2015)

Excitation and emission spectra of purified GPFUV variants. a Purified wtGFPUV and deletion mutants, resolved on 12 % SDS-PAGE. M, marker. Lane 1 to 6, wtGFPUV, C48Δ, P75Δ, P75/D76➔H, E172Δ and S175/V176➔F, respectively. b Normalized excitation spectra. c Normalized emission spectra. Note that the spectra of these variants are largely overlapped
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4513630&req=5

Fig4: Excitation and emission spectra of purified GPFUV variants. a Purified wtGFPUV and deletion mutants, resolved on 12 % SDS-PAGE. M, marker. Lane 1 to 6, wtGFPUV, C48Δ, P75Δ, P75/D76➔H, E172Δ and S175/V176➔F, respectively. b Normalized excitation spectra. c Normalized emission spectra. Note that the spectra of these variants are largely overlapped
Mentions: Deletion mutants C48Δ, P75/D76➔H, P75Δ, E172Δ, S175/V176➔F were purified to more than 95 % homogeneity (Fig. 4a). The emission spectra of all variants under 397 and 475 nm excitation can be superimposed with that of wild-type GFPUV (wtGFPUV) (Fig. 4b), suggesting that the chromophores of these mutants did not have structural alteration. For excitation spectra, two peaks (397 and 475 nm) were observed for all samples. These deletion mutants showed variations in the intensity ratio between the major and minor peaks. For example, P75Δ showed slightly increased minor peak whereas the minor peak in S175/V176➔F mutant was considerably reduced (Fig. 4c). The 395 nm and 475 nm excitation peaks represent neutral and anionic chromophore, respectively [20]. Mutations that alter the ratio of the two chromophore species are frequently found in GFP variants. In one case, enhanced GFP (EGFP) only has a single excitation peak at 475 nm [21] because S65T mutation transform the chromophore to be completely ionized [20]. It is interesting to observe in this study that amino acid deletions, in addition to substitutions, can have impact on the ionization state of chromophore.Fig. 4

Bottom Line: Other approach to reducing structural constraints may include minimizing the structure of GFPs.In this study, we interrogated the structural plasticity of a UV-optimized GFP variant (GFP(UV)) to amino acid deletions, characterized the effects of deletions and explored the feasibility of rescuing the fluorescence of deletion mutants using folding-enhancing mutations.Our results suggested that a "size-minimized" GFP may be developed by iterative incorporation of amino acid deletions, followed by fluorescence rescue with folding-enhancing mutations.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China. liushs@shanghaitech.edu.cn.

ABSTRACT

Background: Green fluorescent protein (GFP) and its derivative fluorescent proteins (FPs) are among the most commonly used reporter systems for studying gene expression and protein interaction in biomedical research. Most commercially available FPs have been optimized for their oligomerization state to prevent potential structural constraints that may interfere with the native function of fused proteins. Other approach to reducing structural constraints may include minimizing the structure of GFPs. Previous studies in an enhanced GFP variant (EGFP) identified a series of deletions that can retain GFP fluorescence. In this study, we interrogated the structural plasticity of a UV-optimized GFP variant (GFP(UV)) to amino acid deletions, characterized the effects of deletions and explored the feasibility of rescuing the fluorescence of deletion mutants using folding-enhancing mutations.

Methods: Transposon mutagenesis was used to screen amino acid deletions in GFP that led to fluorescent and nonfluorescent phenotypes. The fluorescent GFP mutants were characterized for their whole-cell fluorescence and fraction soluble. Fluorescent GFP mutants with internal deletions were purified and characterized for their spectral and folding properties. Folding-ehancing mutations were introduced to deletion mutants to rescue their compromised fluorescence.

Results: We identified twelve amino acid deletions that can retain the fluorescence of GFP(UV). Seven of these deletions are either at the N- or C- terminus, while the other five are located at internal helices or strands. Further analysis suggested that the five internal deletions diminished the efficiency of protein folding and chromophore maturation. Protein expression under hypothermic condition or incorporation of folding-enhancing mutations could rescue the compromised fluorescence of deletion mutants. In addition, we generated dual deletion mutants that can retain GFP fluorescence.

Conclusion: Our results suggested that a "size-minimized" GFP may be developed by iterative incorporation of amino acid deletions, followed by fluorescence rescue with folding-enhancing mutations.

No MeSH data available.


Related in: MedlinePlus