Chapter 5 - Fluorescent Protein Applications in Microscopy
Section snippets
The Identification of Green Fluorescent Protein
The isolation of green fluorescent protein (GFP) was first described by Shimomura, Johnson, & Saiga, 1962 as a footnote to their studies about the aequorin protein from the jellyfish Aequorea aequorea. A. aequorea normally emits a greenish luminescence from the light organs around the rim of the jellyfish. During the isolation of the luminescence system of Aequorea, Shimomura and colleagues noted that the luminescence from aequorin was blue rather than the green luminescence of the intact
Formation of the GFP Chromophore
The primary amino acid sequence of GFP is sufficient to direct the formation of the functional chromophore. Heim, Prasher & Tsien, 1994 first proposed a reaction scheme to explain how GFP might spontaneously mature into a fluorescent protein (FP; Fig. 5.2). Four key steps were proposed to control chromophore formation: (1) the folding of the GFP protein, (2) the cyclization and dehydration of the peptide backbone between the amide nitrogen of Gly67 and the carbonyl of Ser65, (3) the oxidation
The Structure of GFP
The crystal structure of GFP revealed the importance of the entire GFP protein to the fluorescence of the chromophore. GFP is formed by an 11-stranded β-barrel that folds into a structure that has been termed as β-can (Fig. 5.3; Ormo et al., 1996, Yang et al., 1996). The chromophore is completely buried in the center of the β-can structure, protected from solvent quenching effects. Folding of the GFP protein into the β-can structure prevents access of other proteins to the chromophore region;
Mutagenesis to Alter the Properties of GFP
The thermosensitivity, slow maturation rate, and complex absorbance spectrum of Aequorea GFP were some of the factors that prompted multiple research groups to begin mutagenizing GFP to alter its functional properties. Initial mutagenesis studies on GFP were directed at improving its folding and expression at 37 °C and at altering its excitation and emission spectrum (Cormack et al., 1996, Crameri et al., 1996, Delagrave et al., 1995, Heim et al., 1995, Heim et al., 1994, Siemering et al., 1996
Imaging FPs
There are several simple factors to consider and control when planning an imaging experiment that will dramatically improve the ability to detect FPs in fixed or live specimens. Most of these factors are just as applicable to imaging conventional fluorophores as they are to FPs and thus can be considered as general guidelines for fluorescence imaging. The challenge with any live cell experiment is to image the specimen over a long enough time frame to extract meaningful data. This is almost
Applications of FP Imaging
The development of FP variants that possess diverse properties such as spectral differences, sensitivity to environmental factors, and photoswitchable responses to light has opened many new avenues in live cell imaging. Here, we discuss a few examples of microscopy-based FP applications to investigate cell biological questions.
Conclusion
The examples described earlier represent just a few of the many applications of FP imaging used to understand the dynamics of proteins in cells and to monitor the intercellular environment. Additional applications whose descriptions are outside the scope of this chapter include FP-based sensors of cell cycle state (Sakaue-Sawano et al., 2008), optical control of protein activity (Zhou, Chung, Lam & Lin, 2012), all-optical writing with a photoswitchable GFP (Grotjohann et al., 2011), and many
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Cited by (6)
Light-Emitting Probes for Labeling Peptides
2020, Cell Reports Physical ScienceCitation Excerpt :Other self-labeling fluorescent proteins (FPs), such as SNAP-tag and HaloTag, can be conjugated to peptides or proteins to investigate how they associate with cell membranes, or can even be directly coupled to living cells.23,24 However, wild-type FPs are usually found in their oligomeric form, showing higher toxicity and decreased ability to interact with their target biomolecules (e.g., proteins, peptides), leading to mistargeting and lower selective binding, thus requiring artificial monomerization, and consequent loss of brightness and stability.23 Moreover, FPs are large and hydrophobic (Figure 3), and when they are incorporated into small target molecules such as peptides, they can cause undesirable aggregation and changes in physicochemical properties and biological activity.25,26
Live visualization and quantification of pathway signaling with dual fluorescent and bioluminescent reporters
2014, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Moreover, both immune-based and biochemical assays typically use cell lysates, thus limiting their capability to study the dynamics of signal transduction in living cells [10]. High-throughput, image-based cell assays have emerged as alternative approach for monitoring molecular events [11–13]. For example, signaling molecule such as NF-κB can be fused with imageable reporters such as green fluorescent protein (GFP) or red fluorescent protein (RFP).
Determining absolute protein numbers by quantitative fluorescence microscopy
2014, Methods in Cell BiologyCitation Excerpt :This can be most easily achieved using a genetically encoded fluorophore that is both bright and stable (Douglass & Vale, 2008; Johnson & Straight, 2013; Xia, Li, & Fang, 2013). Imaging parameters should minimize sample photobleaching, and all methods discussed are very sensitive to loss of signal intensity due to unintended photobleaching during image acquisition (Coffman & Wu, 2012; Johnson & Straight, 2013). The detailed protocol that follows includes specific guidelines for optimization of image acquisition.
Designing a rigorous microscopy experiment: Validating methods and avoiding bias
2019, Journal of Cell BiologyEvaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants
2018, Journal of Chemical Technology and Biotechnology