Spectral Versatility of Single Reef Coral Fluorescent Proteins Detected by Spectrally‐Resolved Single Molecule Spectroscopy

Abstract

We use spectrally-resolved room temperature single molecule spectroscopy to yield insights into the occurrence and dynamics of spectral forms of single tetramers of DsRed and its variants DsRed2, Fluorescent Timer, DsRed_N42H and AG4. The red-emitting chromophore in DsRed and all studied variants readily converts into a high quantum efficiency super-red emitting form. We propose the existence of two super-red forms of different quantum efficiencies. The observed emission from the green-emitting chromophore is consistent with bulk spectroscopy. We further observe distinct new spectral forms from each variant, which we attribute to a photoinduced chemical reaction leading to a truncated form of the red-emitting chromophore analogous to the chromophore in the visible fluorescent protein zFP538. Our results have implications for the accurate interpretation of biological and biochemical processes illuminated by fluorescent proteins as well as for choosing appropriate experimental configurations.

Single-molecule spectroscopy of fluorescent proteins

“…These three types of spectrum were found for all sampled DsRed variants spanning emission colour from green over orange to red. The frequency of occurrence of the respective spectra varied drastically and was consistent with the bulk spectra [136] Analysis of the emission maximum positions of all single-oligomer spectra of all variants showed that the emission maximum positions of the green-emitting chromophores are distributed around ∼503 nm, consistent with bulk spectroscopy (Fig. 8).…”

“…8). In this , and AG4 show one main distribution of maximum positions whereas the DsRed_N42H variant shows two distinct distributions originating from the red-emitting and green-emitting chromophores, respectively [136]. The emission from the red-emitting chromophore is systematically red-shifted compared with the bulk emission; this is attributed to the rapid (<1 s) formation of a super-red form of the chromophore.…”

“…From Ref. [136] wavelength region the emission cannot be attributed to the main emitting forms. The percentage of tetramers showing this rare form of emission is generally low, but not negligible (DsRed ∼3%, Fluorescent Timer ∼1%, DsRed2 ∼6%, AG4 ∼2%, and DsRed_N42H ∼2%).…”

“…The use of time-resolved measurements and the variation of excitation wavelength further confirmed the complex photophysical behaviour of DsRed that has to be taken into account when DsRed is used to illuminate biological questions. A systematic study of the emission spectra of different DsRed mutants at the single-tetramer level further highlighted the spectral complexity of this group of proteins [136]. Amino acid substitutions in these variants (DsRed2: Arg2Ala, Lys5Glu, Lys9Thr, Val105Ala, Ile161Thr, Ser197Ala; Fluorescent Timer: Val105Ala, Ser197Thr; DsRed_N42H: Asn42His; AG4: Val71Met, Val105Ala, Ser197Thr) yield proteins with altered spectral and maturation properties.…”

“…500 600 500 600 500 600 500 600 λ max = 570 nm λ max = 601 nm intensity / u.u. λ / nm λ / nm λ / nm The spectral series shows the transition between the predominant super-red form and a rare spectral form with emission maximum at 570 nm via a dark state [136] Because the tetramer is composed of a layered dimer of dimers, in principle each monomer has two direct neighbours, one in its layer and one in the other layer of the tetramer. The transfer efficiency between a monomer and its direct neighbour in the other layer of the tetramer is found to be very high (∼96%), whereas the transfer efficiency to the other direct neighbour within its layer is found to be a moderate (75%).…”