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Abstract
A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.
Highlights
Fluorescent reporters are useful for the study of protein dynamics in a live cell, because they can inform on the conformational dynamics of a protein
Post-translational chemical labeling with compact fluorescent dyes through reactive side-chains (Cys, Lys) or with bio-orthogonal labeling allows for the use of more diverse fluorophores and experimental applications (Mannuzzu et al, 1996; Cha and Bezanilla, 1997; Priest et al, 2015; Debets et al, 2013)
A method is presented for the cellular encoding of single-molecule fluorophores as non-canonical amino acids based on widely employed organic Cy dyes for single-molecule studies (Figure 1)
Summary
Fluorescent reporters are useful for the study of protein dynamics in a live cell, because they can inform on the conformational dynamics of a protein. A common strategy to incorporate fluorophores into target proteins is the fusion to fluorescent proteins, such as GFP or one of its spectral variants (Miranda et al, 2013; Zachariassen et al, 2016) This approach has the built-in convenience of encoding but their positioning within the target protein can be limited by their large size, which may impact protein function or trafficking. Cys- or Lys-labeling in live cells is limited to extracellular residues and often results in a substantial background signal due to native reactive residues of the cell This shortcoming is further limiting when labelling sidechains in eukaryotic membrane proteins which can have many ‘background’ cysteine residues, that when mutated can lower expression profiles or have functional consequences. Of the available encodable fluorophores, most are excited by near UV light, a spectroscopic property that leads to competing cellular fluorescence, and have limited, if any, utility for single-molecule studies e.g. they have very short fluorescence lifetimes
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