Abstract
BackgroundIn the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra.ResultsOur results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His). Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP). To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins.ConclusionBased on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mTFP1. With respect to live cell imaging applications, the mTFP1-derived mWasabi should be particularly useful in two-color imaging in conjunction with a Sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue FP donor. In all fusions attempted, both mTFP1 and mWasabi give patterns of fluorescent localization indistinguishable from that of well-established avGFP variants.
Highlights
In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein, the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research
CFihgruormeo1phore structures of mTFP1 and its hue-shifted variants Chromophore structures of mTFP1 and its hue-shifted variants. (A) The chromophore structure shared by enhanced avGFP (EGFP), mTFP1 and mWasabi. (B) The chromophore structure shared by enhanced CFP (ECFP) and the mTFP1-Y67W variant. (C) The chromophore structure shared by EBFP and the mTFP1-Y67H variant
Atoms labeled 'W' are ordered water molecules. (B) The chromophore environment of amFP486 showing the residues that are structurally aligned with the residues represented in (A) (PDB code 2A46) [20]. (C) The chromophore environment of Aequorea victoria green fluorescent protein (avGFP)-S65T showing the residues that structurally align with those represented in (A) (PDB code 1EMA) [26]. avGFP-S65T and EGFP differ only by the Phe64Leu mutation which does not significantly modify the conformation of any residues shown in this figure
Summary
In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. Soon after the first demonstrations of functional expression of the gene encoding avGFP in organisms other than jellyfish [3,4], published reports of the use of fluorescent proteins (FPs) for microscopy applications 'took off' [5]. One important driving force behind the ever-increasing popularity of FPs is the fact that researchers continue to create FPs with wavelength-shifted absorbance and/or emission wavelengths and/or improved or novel properties (for example, increased brightness, improved photostability or photoactivation) [6,7]. FPs with novel properties can inspire the development of entirely new applications that would otherwise be impractical or even impossible. This has certainly been the case with photoactivatable FPs that have enabled cellular imaging at resolutions beyond the diffraction limit [8]
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