We have demonstrated a novel scheme for applying photostability as a selection criterion during directed evolution of fluorescent proteins. Using a high-intensity light source, we are able to photobleach entire 10 cm plates of bacteria expressing the fluorescent proteins of interest and select those that maintain the most brightness. This approach allowed us to screen libraries containing up to 100,000 clones reliably with no observed false-positive hits. While the precise kinetics of photobleaching for a given fluorescent protein are strongly dependent on illumination intensity and temporal regimen, we have found that improvements in photostability at ~0.1 W/cm2
usually qualitatively predict improved performance under typical conditions for wide-field and laser scanning microscopy. The exceptions are mApple's reversible photoswitching (Supplementary Note 1
online) and tdTomato's poor performance under laser scanning confocal illumination (). Also, while our screen utilized bacteria to express fluorescent protein libraries, all proteins produced from these studies behaved similarly when later tested in purified form or expressed in mammalian cells, consistent with our previous experience.
Photobleaching using an array of LEDs was performed during the evolution of mTFP1 to select against unacceptable photolability or photoswitching, resulting in a protein with a bleaching half-time 110 seconds12
. Our work applies photostability as a primary criterion to improve multiple fluorescent proteins, and successfully demonstrates that high photostability is a selectable phenotype. Moreover, a solar simulator benefits from the strong mercury lines at 546, 577, and 579 nm and allows greater flexibility in the choice of excitation wavelength than would be possible with LEDs.
While it is difficult to draw strong conclusions from the photostability mutations in mOrange2, specific regions proximal to the chromophore appear to influence the modes of photobleaching it is able to undergo. DsRed, when illuminated by a 532nm pulsed laser, undergoes decarboxylation of Glu215, as well as cis
isomerization of the chromophore19
. Such chromophore isomerization has been implicated in the photoswitching behavior of Kindling fluorescent protein (KFP)20, 21
and Dronpa5, 22
, as well as predecessors to mTFP1 (refs. 12
). Decarboxylation of the corresponding glutamate (position 222) in Aequorea
GFP also leads to changes in optical properties24-26
. However, our observation that oxidation plays a large role in mOrange, TagRFP, and TagRFP-T photobleaching suggests that chromophore isomerization and Glu215 decarboxylation may play only a minor role for such proteins under normoxic conditions. Additionally, we found no evidence by mass spectrometry that photobleaching using the solar simulator led to any detectable decarboxylation of Glu215 in mOrange (data not shown). Under some conditions mOrange2 shows an initial photoactivation of about 5% () before bleaching takes over. At present we have no molecular explanation for this minor effect or the reversible photoswitching shared by most fluorescent proteins (see Results
and Supplementary Note 3
For mRFP1 variants, we have clearly observed the importance of residue 163 in influencing photostability (see Supplementary Note 1
online), but have also seen somewhat context-specific effects of 163 and surrounding residues on different wavelength-shifted variants. This region, composed of residues 64, 97, 99, and 163, appears to be important in determining photostability. However, of these, only residue 163 is in direct contact with the chromophore. It may be that the mutations Q64H and F99Y together lead to a rearrangement of the other side chains in the vicinity of the chromophore so as to hinder a critical oxidation that leads to loss of fluorescence.
Discrepancies in tubulin and connexin localization (see Supplementary Note 2
online) when fused to mOrange2 versus
mKO or tdTomato can probably be attributed to the three-dimensional structure of the fluorescent protein and potential steric hindrance in the fusions. mOrange2 contains extended N- and C- termini derived from EGFP to improve performance in fusions, whereas the much shorter protein, mKO (236 vs. 218 amino acids, respectively), may experience steric interferences that lead to poorer performance in similar fusions. The fused dimeric character of tdTomato effectively doubles its size compared to the monomeric orange fluorescent proteins, so steric hindrance is the most likely culprit in preventing tubulin localization. For most fusions, however, we observed little or no difference in performance between mOrange2 and mKO, suggesting that many proteins are more tolerant of fusion partners than tubulin or connexins.
We have shown that our photostability selection method may be applied to TagRFP, which, though it already possesses reasonably good photostability, was still amenable to improvements. From a saturation-mutagenesis library of two chromophore-proximal residues (consisting of 400 independent clones), we selected a single clone with substantially enhanced photostability. The selected mutant, TagRFP-T, should prove to be a very useful addition to the fluorescent protein arsenal, as it is the most photostable monomeric fluorescent protein of any color yet described under both arc lamp and confocal laser illumination.
As the applications of genetically encoded fluorescent markers continue to diversify and become more complex, the demand for greater photostability than is available in current fluorescent proteins has likewise continued to grow. We have expanded existing directed evolution approaches by utilizing medium-throughput photostability selection. We expect this novel screening method to be applicable to any of the large number of existing fluorescent proteins, and, with modifications, to be useful in selecting for more efficient photoconvertible and photoswitchable fluorescent proteins as well3, 5, 10, 20, 27-31
. Possible enhancements to this selection technique could include time-lapse imaging of bacterial plates during bleaching to enable direct selection for kinetics (independent of absolute brightness) and the use of higher intensity illumination from other light sources (such as lasers) during screening to select for or against non-linear photobleaching behavior. Ideally, a selection scheme that allows true simulation of microscopic imaging light intensities while maintaining a medium- to high-throughput should allow selection of fluorescent proteins with the most beneficial properties for imaging applications.