The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.
To visualize Ca 2؉ -dependent protein-protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini. The cpGFP was fused to calmodulin and its target peptide, M13. The chimeric protein, which we have named ''pericam,'' was fluorescent and its spectral properties changed reversibly with the amount of Ca 2؉ , probably because of the interaction between calmodulin and M13 leading to an alteration of the environment surrounding the chromophore. Three types of pericam were obtained by mutating several amino acids adjacent to the chromophore. Of these, ''flash-pericam'' became brighter with Ca 2؉ , whereas ''inverse-pericam'' dimmed. On the other hand, ''ratiometric-pericam'' had an excitation wavelength changing in a Ca 2؉ -dependent manner. All of the pericams expressed in HeLa cells were able to monitor free Ca 2؉ dynamics, such as Ca 2؉ oscillations in the cytosol and the nucleus. Ca 2؉ imaging using high-speed confocal line-scanning microscopy and a flashpericam allowed to detect the free propagation of Ca 2؉ ions across the nuclear envelope. Then, free Ca 2؉ concentrations in the nucleus and mitochondria were simultaneously measured by using ratiometric-pericams having appropriate localization signals, revealing that extra-mitochondrial Ca 2؉ transients caused rapid changes in the concentration of mitochondrial Ca 2؉ . Finally, a ''split-pericam'' was made by deleting the linker in the flash-pericam. The Ca 2؉ -dependent interaction between calmodulin and M13 in HeLa cells was monitored by the association of the two halves of GFP, neither of which was fluorescent by itself.A pplication of green fluorescent protein (GFP)-based fluorescence resonance energy transfer (FRET) allows to visualize protein heteromerization and conformational changes in single living cells (1). Whereas FRET utilizes two GFP mutants having different colors, we have developed single GFPs sensitive to physiologically relevant substrates (e.g., calcium ions in the present study).Wild-type GFP (WT-GFP) has a bimodal absorption spectrum with two peak maxima, at 395 and 475 nm, corresponding to the protonated and the deprotonated states of the chromophore, respectively (2). The ionization state is modulated by a proton network, comprising an intricate network of polar interactions between the chromophore and several surrounding amino acids. In contrast to WT-GFP, the chromophore of most GFP variants titrates with single pKa values, indicating that the internal proton equilibrium has been disrupted as a result of an external manipulation (3). One of the variants is the yellow fluorescent protein (YFP). It has a T203Y substitution that is responsible for the red-shift emission at 528 nm (2). It has been predicted that the tyrosine introduced at position 203 would be involved in a -stacking interaction with the chromophore (4), which has been demonstrated by ...
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