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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

Efficiency of chromophore maturation. Left panel, absorbance of wtGFPUV and variants under base-denatured condition. Right panel, efficiency of chromophore maturation, derived from the absorbance data. Three measurement replicates were performed. Standard deviation is within instrumental error (2 %) and thus not shown in the figure
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Fig6: Efficiency of chromophore maturation. Left panel, absorbance of wtGFPUV and variants under base-denatured condition. Right panel, efficiency of chromophore maturation, derived from the absorbance data. Three measurement replicates were performed. Standard deviation is within instrumental error (2 %) and thus not shown in the figure

Mentions: Instructed by these results, we then analyzed the efficiency of chromophore maturation of deletion mutants. In this experiment, we denatured 7.0 μM protein of each sample and determined their absorbance at 450 nm (Fig. 6). As the extinction coefficient of matured chromophore at 450 nm has been determined in previous study [25], the concentration of matured chromophore can be calculated using Beer’s law. It is evident that all deletions reduced the efficiency of chromophore maturation (Fig. 6). It is worth mentioning that the protein samples used for this experiment was purified at 20 °C. Previous analysis of the whole-cell lysis showed that the five mutant with internal deletions displayed only trace fluorescence but had considerable amount of soluble proteins when expressed at 30 °C. According to the analysis of chromophore, this discrepancy is likely attributed to the impaired chromophore maturation of GFP proteins present in the soluble fraction.Fig. 6


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)

Efficiency of chromophore maturation. Left panel, absorbance of wtGFPUV and variants under base-denatured condition. Right panel, efficiency of chromophore maturation, derived from the absorbance data. Three measurement replicates were performed. Standard deviation is within instrumental error (2 %) and thus not shown in the figure
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Efficiency of chromophore maturation. Left panel, absorbance of wtGFPUV and variants under base-denatured condition. Right panel, efficiency of chromophore maturation, derived from the absorbance data. Three measurement replicates were performed. Standard deviation is within instrumental error (2 %) and thus not shown in the figure
Mentions: Instructed by these results, we then analyzed the efficiency of chromophore maturation of deletion mutants. In this experiment, we denatured 7.0 μM protein of each sample and determined their absorbance at 450 nm (Fig. 6). As the extinction coefficient of matured chromophore at 450 nm has been determined in previous study [25], the concentration of matured chromophore can be calculated using Beer’s law. It is evident that all deletions reduced the efficiency of chromophore maturation (Fig. 6). It is worth mentioning that the protein samples used for this experiment was purified at 20 °C. Previous analysis of the whole-cell lysis showed that the five mutant with internal deletions displayed only trace fluorescence but had considerable amount of soluble proteins when expressed at 30 °C. According to the analysis of chromophore, this discrepancy is likely attributed to the impaired chromophore maturation of GFP proteins present in the soluble fraction.Fig. 6

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