Evaluation of Genetically Encoded Chemical Tags as Orthogonal Fluorophore Labeling Tools for Single-Molecule FRET Applications.

Fluorescence resonance energy transfer (FRET) is a superb technique for measuring conformational changes of proteins on the single molecule level (smFRET) in real time. It requires introducing a donor and acceptor fluorophore pair at specific locations on the protein molecule of interest, which has often been a challenging task. By using two different self-labeling chemical tags, such as Halo-, TMP-, SNAP- and CLIP-tags, orthogonal labeling may be achieved rapidly and reliably. However, these comparatively large tags add extra distance and flexibility between the desired labeling location on the protein and the fluorophore position, which may affect the results. To systematically characterize chemical tags for smFRET measurement applications, we took the SNAP-tag/CLIP-tag combination as a model system and fused a flexible unstructured peptide, rigid polyproline peptides of various lengths, and the calcium sensor protein calmodulin between the tags. We could reliably identify length variations as small as four residues in the polyproline peptide. In the calmodulin system, the added length introduced by these tags was even beneficial for revealing subtle conformational changes upon variation of the buffer conditions. This approach opens up new possibilities for studying conformational dynamics, especially in large protein systems that are difficult to specifically conjugate with fluorophores.

Intracellular thermometry by using fluorescent gold nanoclusters.

The "gold standard" for nanothermometry: The application of ultrasmall, near-IR-emitting fluorescent gold nanoclusters (AuNCs) for temperature sensing has been explored. AuNC-based fluorescent nanothermometry features excellent thermal sensitivity and simultaneous temperature sensing and imaging in HeLa cells.

One-pot synthesis of near-infrared fluorescent gold clusters for cellular fluorescence lifetime imaging.

A facile strategy to synthesize water-soluble fluorescent gold nanoclusters (Au NCs) stabilized with the bidentate ligand dihydrolipoic acid (DHLA) is reported. The DHLA-capped Au NCs are characterized by UV-vis absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy. The Au NCs possess many attractive features including ultrasmall size, bright near-infrared luminescence, high colloidal stability, and good biocompatibility, making them promising imaging agents for biomedical and cellular imaging applications. Moreover, their long fluorescence lifetime (>100 ns) makes them attractive as labels in fluorescence lifetime imaging (FLIM) applications. As an example, the internalization of Au NCs by live HeLa cells is visualized using the FLIM technique.