Highly photostable fluorescent labeling of proteins in live cells using exchangeable coiled coils heterodimerization

Abstract

Fluorescent proteins are commonly used to label target proteins in live cells. However, the conventional approach based on covalent fusion of targeted proteins with fluorescent protein probes is limited by the slow rate of fluorophore maturation and irretrievable loss of fluorescence due to photobleaching. Here we report a genetically encoded protein labeling system utilizing transient interactions of small, 21-28 residues-long helical protein tags (K/E coils, KEC). In this system, a protein of interest, covalently tagged with a single coil, is visualized through binding to a cytoplasmic fluorescent protein carrying a complementary coil. The reversible heterodimerization of KECs, whose affinity can be tuned in a broad concentration range from nanomolar to micromolar, allows continuous exchange and replenishment of the tag bound to a targeted protein with the entire cytosolic pool of soluble fluorescent coils. We found that, under conditions of partial illumination of living cells, the photostability of labeling with KECs exceeds that of covalently fused fluorescent probes by approximately one order of magnitude. Similarly, single molecule localization microscopy with KECs provided higher labeling density and allowed a *

LIVE-PAINT allows super-resolution microscopy inside living cells using reversible peptide-protein interactions

“…Also, LIVE-PAINT is extendible to concurrent tagging of multiple proteins. Recently, interacting charged coiled coil pairs have been used to label proteins to perform PALM imaging in live mammalian cells 32 . This work provides a valuable independent validation of our approach.…”

Studying SARS-CoV-2 with Fluorescence Microscopy

“…In a new method, called KECs (K/E coils), the fluorescent protein and the target protein are not part of a single polypeptide chain, but are linked through reversible specific heterodimerization of alpha-helices (one helix is fused with a fluorescent protein, the other with the target protein, Figure 4 B) [ 66 ]. As a result, a pre-expressed and matured fluorescent protein in the cytosol immediately binds to the newly synthesized target protein and reveals its localization (the method is applicable only for proteins with distinct localization, for example, cytoskeletal and membrane proteins).…”

PAINT using proteins: A new brush for super‐resolution artists

“…Perfilov et al showed that different versions of the E 3 /K 3 peptides (containing point mutations) could be used to perform super-resolution imaging in live cells. 3 In this example, they used a peptide attached to a photoconvertible fluorescent protein, and used photoactivated localization microscopy (PALM) to obtain data for a super-resolution image. Although a peptide-protein interaction is used in this work, it differs from the work of Eklund et al 1 and Oi et al, 2 in not employing a PAINT approach to data acquisition.…”

“…It should not be too tight, because the PAINT approach relies on transient interactions and exchange of the bound state with the unbound pool. Operationally, a dissociation constant of about 1 μM is desirable, 1,3,9 although the on‐rate and off‐rate of binding is more important than the K d .…”

“…Recent papers describe the successful use of transient peptide–protein interactions to perform super‐resolution microscopy, 1–3 in particular as a new way to implement the method known as points accumulation for imaging in nanoscale topography (PAINT) 4 (Figure 1).…”