Abstract
In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.
Highlights
® FP variants are less bright and photostable than synthetic fluorophores (e.g., AlexaFluor dyes) and are known to show large variations in brightness due to differential chromophore maturation[2,12]
A linear peptide model with no super-secondary interaction was generated by extending the PEP-FOLD model (Fig. 1b) with Adaptive Steered Molecular Dynamics (ASMD)
The arsenical hairpin derivative of fluorescein, fluorescein arsenical hairpin (FlAsH), offers a highly specific labelling strategy of proteins via a small tetracysteine motif (FCM) which can be inserted in solvent accessible regions, typically looped or disordered regions
Summary
® FP variants are less bright and photostable than synthetic fluorophores (e.g., AlexaFluor dyes) and are known to show large variations in brightness due to differential chromophore maturation[2,12]. A series of tetracysteine motif variants, have been used to study a wide range of biological systems, ranging from pathogenic effectors in bacterial cells to β-tubulin in yeast cells[18], and even mammalian G protein-coupled receptors (GPCRs)[19,20,21] This method of labelling has been used in combination with FP-variants of the POI, in order to obtain dynamic information regarding changes in protein conformation or protein-protein association by monitoring changes in FRET in live cells[19,22]. The photophysical properties of bisarsenical fluorophores have not been studied in detail and their suitability as single-molecule (sm) fluorescent probes has yet to be assessed This information will be useful for designing smFRET experiments using these versatile fluorescent tags, and for disentangling protein fluctuations from photophysics dynamics (i.e., photoblinking) in fluorescence quenching experiments (e.g., FCS, lifetime)[23]. For bisarsenical fluorophores the relevant photophysics has not been studied well, and the potential use of these probes for PET studies still has to be assessed
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