Rapid labeling of intracellular His-tagged proteins in living cells

Abstract

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA–based probes. Unfortunately, previous Ni-NTA–based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

Integrative approach for the analysis of the proteome-wide response to bismuth drugs in Helicobacter pylori

“…2A), by reacting equimolar amounts of Bi 3+ (as bismuth nitrate) with a fluorescent ligand NTA-AC 18 in a buffered aqueous solution, where the formation of Bi 3+ -TRACER was confirmed by ESI-MS (Fig. S1†).…”

Self-assembly of c-myc DNA promoted by a single enantiomer ruthenium complex as a potential nuclear targeting gene carrier

“…3)42. Thus, the FAM (green)-tagged c-myc G-quadruplex DNA was used to indicate the location of DNA in cells.…”

Multiplexed imaging of intracellular protein networks

“…Similarly, Halo‐tag binds synthetic dyes having a chloroalkane linker . Furthermore, cell‐permeable fluorescent probes that bind His‐tag were recently developed . These labeling systems are orthogonal (i.e., do not crosstalk with each other) and therefore can be combined together for multiplexed imaging in live cells .…”