moxDendra2: an inert photoswitchable protein for oxidizing environments

Abstract

Fluorescent proteins (FPs) that can be optically highlighted, enable PALM super-resolution microscopy and pulse-chase experiments of cellular molecules. Most FPs were evolved in and for the cytoplasm and, therefore, may fold incorrectly in subcellular organelles. Here, we describe the first monomeric photoswitchable from green to red bright FP adapted for oxidizing environments.

moxMaple3: a Photoswitchable Fluorescent Protein for PALM and Protein Highlighting in Oxidizing Cellular Environments

“…It was not obvious a priori that the cysteines of mMaple3 could be mutated without disrupting the desirable properties of mMaple3. Similar efforts for EGFP family members often resulted in dark proteins and our previous efforts with the development of moxEos3.2 and moxDendra2 9 had revealed that the cysteine positions were highly sensitive even to seemingly conservative small amino acid substitutions. Serine was never tolerated and an alanine or valine could produce dramatic differences in apparent brightness 3 , 5 , 18 .…”

“…Consequently, these post-translational modifications and environmental effects can lead to FP misfolding, dark FPs, and mislocalization 3 – 6 . Recently, our groups successfully engineered FPs into “mox” (monomeric nonoxidizing or disulphide bonding) forms to improve their suitability for a wide variety of eukaryotic cellular compartments 5 , 9 . Here we turn our attention to develop an improved photoswitchable FP to optically highlight fusion proteins to study their fate and for single-molecule-based photoactivation localization microscopy PALM super-resolution imaging 10 – 12 .…”

A Large Stokes Shift Fluorescent Protein Constructed from the Fusion of Red Fluorescent mCherry and Far‐Red Fluorescent BDFP1.6

“…The labeled fusion proteins MTS:BDFP2.0 and NLS:BDFP1.1 can be coexpressed correctly in HeLa cells, and interference between the two protein markers can be greatly reduced because of a large gap between the two excitation lasers (Figure ). These results indicate that BDFP2.0 can be correctly folded in cells and the resulting chromophores are highly stable, which does not alter the localization, function, and behavior of a tagged fusion protein …”

“…The size‐exclusion chromatography results indicate that BDFP2.0 may have two aggregation states, that is, the majority is a dimeric state (75 kDa) and the minority is a monomeric state (45 kDa) in solution (Figure S3 B). To test this, we fused BDFP2.0 to the monomericity reporter CytERM, which expressed the reporter in HeLa cells and imaged cells for evidence of membrane accumulation by oligomeric proteins . The results of the CytERM assay also suggested that BDFP2.0 might exist as a dimer in vivo.…”

“…If BDFP1.6 was fused with mCherry, the resulting BDFP2.0 could be more rapidly maturated than that of mCherry (Figure S2 C). Meanwhile, the increased brightness may have a noticeable impact on performance if BDFP2.0 is expressed in an oxidizing environment, such as mitochondria and Gram‐negative E. coli periplasm . Based on the advantage of the large Stokes shift resulting from fusion of BDFP1.6 with mCherry, BDFP2.0 may be applied to quantify protein–protein interactions, such as in dual‐color fluorescence cross‐correlation spectroscopy .…”