Bright blue-shifted fluorescent proteins with Cys in the GAF domain engineered from bacterial phytochromes: fluorescence mechanisms and excited-state dynamics

Abstract

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.

How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells

“…Because the BphP-derived miRFP670, mIFP and miRFP703 already exhibit the efficient binding of endogenous chromophore, future engineering efforts on these NIR FPs should focus on an increase of their quantum yields, e.g., via reduction of various excited-state deactivation pathways (Hontani et al, 2016). In contrast, the APCα-derived smURFP should be further evolved to substantially enhance its efficiency and specificity to bind endogenous BV chromophore in mammalian cells.…”

Near-Infrared Fluorescent Proteins and Their Applications

“…It was shown that in BphP1 FP, BV could be linked to Cys GAF via C3 1 or C3 2 carbon atoms of the pyr role ring A side chain, both chromophore derivatives being spectrally identical [27]. Two suggestions have been made to explain the high quantum yield of NIR FPs with the fluorescence spectra displaying hypsochromic shift: (i) the chromophore bound to Cys GAF has more rigid structure due to the limited mobility of the pyrrole ring A; (ii) mobility of the ring A does not affect the properties of BV Cys GAF , because in this case, it is not included in the conjugated system of π bonds [47]. Chromophore inter action with dimeric NIR FPs is complicated by inter monomer allosteric effect, with a number of spectral characteristics being sensitive to this effect.…”

Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes

“…BphP1-FP, iRFP670 and iRFP682 proteins, in addition to conserved Cys20 in the PAS domain, contain Cys residue in the conserved SPXH amino acid motif of the GAF domain (Cys253). NIR FPs with this additional Cys are blue-shifted, exhibit higher quantum yield and longer fluorescence lifetimes [ 27 , 109 , 110 ]. It was shown that mutants of such double-Cys NIR FPs, containing a single Cys either in the PAS or in the GAF domains, demonstrate different spectral properties.…”

“…These findings made possible the rational design of brighter and spectrally distinct NIR FPs and were successfully applied to engineer spectrally distinct monomeric miRFPs [ 35 ]. In BphP-derived NIR FPs, the covalent binding of BV to Cys residue in the GAF domain via C3 1 and C3 2 chromophore atom leads to restricting ring A motion, resulting in a substantial increase of quantum yield [ 109 ]. Interestingly, these NIR FPs bind Cys in the GAF domain similarly to CBCRs, which also bind chromophore through C3 1 carbon of ring A.…”

“…The latter one limits the fluorescence quantum yield of NIR FPs. Studies of several NIR FPs suggested that their C15=C16 photoisomerization was completely inhibited, but quantum yield was still rather low [ 109 , 114 , 115 ]. Hence, to improve quantum yield of BphP-based NIR FPs it is necessary to minimize non-radiative decay [ 109 , 114 ].…”