Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are Ϸ25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.T he red fluorescent protein cloned from Discosoma coral (DsRed or drFP583) (1) holds great promise for biotechnology and cell biology as a spectrally distinct companion or substitute for the green fluorescent protein (GFP) from the Aequorea jellyfish (2). GFP and its blue, cyan, and yellow variants have found widespread use as genetically encoded indicators for tracking gene expression and protein localization and as donor͞ acceptor pairs for f luorescence resonance energy transfer (FRET). Extending the spectrum of available colors to red wavelengths would provide a distinct label for multicolor tracking of fusion proteins, and together with GFP (or a suitable variant) would provide a FRET donor͞acceptor pair that should be superior to the currently preferred cyan͞yellow pair (3). However, the evolution of DsRed from a scientific curiosity to a generally applicable and robust tool has been hampered by several critical problems, including a slow and incomplete maturation and obligate tetramerization (4). Most previous attempts to address the rate and͞or extent of maturation of DsRed (5, 6), including the commercially available DsRed2 (CLONTECH), have provided only modest improvements. However, an engineered variant of DsRed, known as T1 (see Fig. 1A), has recently become available and effectively solved the problem of the slow maturation (7). Another approach to overcoming these shortcomings has been to continue the search for DsRed homologues in sea coral and anemone, an ...

(2) to aromatic amino acids, typically Tyr. The resulting -stacking and increased local polarizability immediately adjacent to the chromophore are believed to be responsible for the ϳ20-nm shift to longer excitation and emission wavelengths (3). However, the changes in internal hydrogen bonding and steric packing also made the fluorescence more vulnerable to photobleaching (4, 5), decolorization by protonation (6 -10), and quenching by many anions (10 -12), of which chloride is the physiologically most relevant. These sensitivities can be exploited for specialized applications such as measuring fluorescence recovery after photobleaching and sensing pH and halide concentrations, but are deleterious for using YFPs either as simple fusion tags or as acceptors for fluorescence resonance energy transfer (FRET). YFPs are becoming very popular in such roles, particularly as partners for cyan fluorescent protein (CFP) mutants of GFP (2, 5, 13-15). CFPs and YFPs are spectroscopically well enough separated to be easily distinguishable in either excitation or emission spectra, yet the emission wavelengths of CFPs and excitation wavelengths of YFPs overlap well enough to make them good partners for FRET. They have largely superseded the initial pairing of blue mutants and improved green forms of GFP (16), because the blue mutants were too dim and photobleachable, and because shorter wavelengths generically excite more autofluorescence and raise more concerns of phototoxicity. Measurements of FRET between CFP and YFP are becoming increasingly common to monitor protein-protein interactions nondestructively in live cells (5,13,17). The potential partners are fused to CFP and YFP, respectively, and coexpressed in cells. Because FRET requires that the CFP and YFP be within a few nanameters of each other, it can detect proximity at molecular dimensions, with 2 orders of magnitude higher spatial resolution than simple co-localization of the two colors.

Many areas of biology and biotechnology have been revolutionized by the ability to label proteins genetically by fusion to the Aequorea green f luorescent protein (GFP). In previous fusions, the GFP has been treated as an indivisible entity, usually appended to the amino or carboxyl terminus of the host protein, occasionally inserted within the host sequence. The tightly interwoven, three-dimensional structure and intricate posttranslational self-modification required for chromophore formation would suggest that major rearrangements or insertions within GFP would prevent f luorescence. However, we now show that several rearrangements of GFPs, in which the amino and carboxyl portions are interchanged and rejoined with a short spacer connecting the original termini, still become f luorescent. These circular permutations have altered pKa values and orientations of the chromophore with respect to a fusion partner. The Aequorea green fluorescent protein (GFP) is a 238-aa, spontaneously fluorescent protein that has become spectacularly popular in molecular and cell biology as a transcriptional reporter, fusion tag, biosensor, or partner for fluorescence resonance energy transfer (FRET) (1). GFP has been fused to a very wide variety of proteins to render them fluorescent, but in almost every case the GFP, even if mutated to change colors or improve folding, simply has been appended to either the amino or carboxyl terminus of the host protein. In a few cases, GFP has been inserted inside the host protein, but even here the GFP-coding sequence has been kept essentially intact (2, 3). Random peptides up to 20 residues in length have been inserted at several locations within loops of GFP, but mostly with deleterious effects on GFP folding and fluorescence (4).GFP is a seemingly monolithic, 11-stranded ␤-barrel that forms a nearly perfect shell around a chromophore spontaneously generated by an unusual multistep pathway involving cyclization and oxidation of residues 65-67 (1). Such a complex maturation process leading to a highly compact and rigid unit would seem very unlikely to tolerate major transpositions and insertions. Nevertheless, we now show that GFP can remain fluorescent despite a variety of circular permutations (5) and insertions of foreign proteins. When the insert itself is a conformationally sensitive receptor, ligand binding can strongly modulate the fluorescence of the GFP. Thus, insertion of receptors into GFP may offer a new strategy for generating genetically encoded indicators for important biochemical and physiological signals. MATERIALS AND METHODSCloning and Gene Construction. Enhanced yellow fluorescent proteins (EYFPs) with peptide insertions replacing Y145 were made by performing two separate PCRs on cDNA encoding EYFP, which is equivalent to GFP with the mutations S65G, V68L, Q69K, S72A, and T203Y, previously termed 10C Q69K (1, 6). The first PCR amplified the 5Ј end of the EYFP cDNA and incorporated a 5Ј BamHI site. The 3Ј primer encoded the hexapeptide linker GGTGEL in place of Y145 a...