How to obtain labeled proteins and what to do with them

Abstract

We review new and established methods for the chemical modification of proteins in living cells and highlight recent applications. The review focuses on tag-mediated protein labeling methods, such as the tetracysteine tag and SNAP-tag, and new developments in this field such as intracellular labeling with lipoic acid ligase. Recent promising advances in the incorporation of unnatural amino acids into proteins are also briefly discussed. We describe new tools using tag-mediated labeling methods including the super-resolution microscopy of tagged proteins, the study of the interactions of proteins and protein domains, the subcellular targeting of synthetic ion sensors, and the generation of new semisynthetic metabolite sensors. We conclude with a view on necessary future developments, with one example being the selective labeling of non-tagged, native proteins in complex protein mixtures. IntroductionChemists are becoming increasingly fascinated with derivatizing proteins by genetically non-encodable synthetic molecules. As discussed in this review, such molecules include fluorescent dyes, chemical crosslinkers, pharmacologically active compounds, and synthetic fluorescent probes for ions such as Ca 2+ . The labeling of proteins with synthetic molecules provides exciting new tools for studying proteins and their function in the cell, and is promising to have a strong impact on drug development. In this review, we will provide a concise overview over established and new methods that provide proteins derivatized with a label, and give illustrative recent examples of their application. For further information, the reader is directed to recently published more exhaustive reviews [1][2][3][4]. The focus of our review lies on covalent tag-based labeling methods, as these allow an efficient and irreversible transfer of labels in mammalian cells. However, we will also comment on unnatural amino acid incorporation as a tool of increasing importance for mammalian cell studies. The discussion of methods and applications is preceded by general considerations on tag-mediated labeling. We will conclude with an outlook on future directions and highlight areas that would profit most from new developments. Tag-mediated labelingAn ideal method for tag-based protein labeling should exhibit the following features: (i) the possibility to introduce any label of choice in one step, (ii) fast and quantitative labeling, (iii) no labeling of non-target proteins, (iv) a small tag to minimize its impact on protein function, (v) the formation of a stable, covalent bond between protein and label, and (vi) no side effects of the reagents used for labeling. Finally, an ideal tag should work in vitro, in complex protein samples, on the cell surface, within the cell and cellular compartments, and in living animals (in vivo), with this order representing an increasing level of difficulty. None of the existing labeling methods fulfils all these requirements. It is notable, however, that the existing methods of tag-mediated labeling can be grouped ...

A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

“…SiR derivatives were shown to possess excellent spectroscopic properties, and in certain cases to be membrane permeable 8 . To achieve selective coupling of SiR derivatives to the proteins of interest, we envisioned exploiting self-labelling protein tags such as SNAP-tag 1 . SNAP-tag fusion proteins can be labelled specifically with molecular probes using benzylguanine (BG) derivatives 10,11 .…”

“…F lexible and specific methods to couple synthetic fluorescent probes to proteins in living cells are established, yet these methods are not generally compatible with the fluorophores best suited for live-cell imaging 1 . Fluorescent labels that are excited and emit in the near-infrared are especially biocompatible, because they avoid the use of light, which may cause phototoxicity or unwanted autofluorescent background 2 .…”

Efficient N‐Terminal Labeling of Proteins by Use of Sortase

“…[3] Each of these methods has its own associated challenges: Selective labeling of a single cysteine residue frequently requires rounds of site-directed mutagenesis to introduce the labeling site and/or remove other cysteine residues, and selective labeling of the N-terminal amino group requires careful control of pH to ensure lysine residues are not also modified. [4] Other modern methods for chemoselective labeling often require the introduction of specific recognition sequences [5] or nonnatural amino acids into the protein to be labeled. [6] In most cases, a substantial excess of the labeling reagent is necessary to ensure complete conversion to the product.…”

Multicolor Single Molecule Tracking of Stochastically Active Synthetic Dyes

“…3 Thus, they offer an interesting alternative to other forms of labeling by fluorescent proteins or ligands. Unlike antibody labeling, they are also compatible with live-cell imaging.…”