Genetically encodable fluorescent protein markers in advanced optical imaging

Optical fluorescence microscopy plays a pivotal role in the exploration of biological structure and dynamics, especially on live specimens. Progress in the field relies, on the one hand, on technical advances in imaging and data processing and, on the other hand, on progress in fluorescent marker technologies. Among these, genetically encodable fluorescent proteins (FPs) are invaluable tools, as they allow facile labeling of live cells, tissues or organisms, as these produce the FP markers all by themselves after introduction of a suitable gene. Here we cover FP markers from the GFP family of proteins as well as tetrapyrrole-binding proteins, which further complement the FP toolbox in important ways. A broad range of FP variants have been endowed, by using protein engineering, with photophysical properties that are essential for specific fluorescence microscopy techniques, notably those offering nanoscale image resolution. We briefly introduce various advanced imaging methods and show how they utilize the distinct properties of the FP markers in exciting imaging applications, with the aim to guide researchers toward the design of powerful imaging experiments that are optimally suited to address their biological questions

Mechanistic Understanding of Protein Corona Formation around Nanoparticles: Old Puzzles and New Insights

Although a wide variety of nanoparticles (NPs) have been engineered for use as disease markers or drug delivery agents, the number of nanomedicines in clinical use has hitherto remained small. A key obstacle in nanomedicine development is the lack of a deep mechanistic understanding of NP interactions in the bio-environment. Here, the focus is on the biomolecular adsorption layer (protein corona), which quickly enshrouds a pristine NP exposed to a biofluid and modifies the way the NP interacts with the bio-environment. After a brief introduction of NPs for nanomedicine, proteins, and their mutual interactions, research aimed at addressing fundamental properties of the protein corona, specifically its mono-/multilayer structure, reversibility and irreversibility, time dependence, as well as its role in NP agglomeration, is critically reviewed. It becomes quite evident that the knowledge of the protein corona is still fragmented, and conflicting results on fundamental issues call for further mechanistic studies. The article concludes with a discussion of future research directions that should be taken to advance the understanding of the protein corona around NPs. This knowledge will provide NP developers with the predictive power to account for these interactions in the design of efficacious nanomedicines

Highly Luminescent Positively Charged Quantum Dots Interacting with Proteins and Cells

We have studied interactions between positively charged MUTAB-stabilized quantum dots (QDs) and model proteins, serum and live cells using fluorescence correlation spectroscopy (FCS), dynamic light scattering (DLS), time-resolved photoluminescence (PL) and live-cell fluorescence imaging. Using human serum albumin (HSA) as a model protein, we measured the growth of a protein adsorption layer (“protein corona”) via time-resolved FCS. Corona formation was characterized by an apparent equilibrium dissociation coefficient, K$_{D}$ ≈ 10 μM. HSA adlayer growth was surprisingly slow (timescale ca. 30 min), in stark contrast to many similar measurements with HSA and other proteins and different NPs. Time-resolved PL data revealed a characteristic quenching behavior depending on the QD surface coverage with HSA. Taken together, we found that MUTAB-QDs initially bind HSA molecules weakly (K$_{D}$ ≈ 700 μM); however, the affinity is enhanced over time, presumably due to proton injection into the MUTAB layer by HSA triggering ligand dissociation. This process was also observed with human blood serum, showing equal kinetics for comparable HSA concentration. Moreover, imaging experiments with cultured human cells (HeLa) revealed that MUTAB-QDs bind to the cell membrane and perforate it. This process is reduced upon pre-adsorption of proteins on the MUTAB-QD surfaces

Exploring the energy landscape of a SAM-I riboswitch

SAM-I riboswitches regulate gene expression through transcription termination upon binding a S-adenosyl-L-methionine (SAM) ligand. In previous work, we characterized the conformational energy landscape of the full-length Bacillus subtilis yitJ SAM-I riboswitch as a function of Mg$^{2+}$ and SAM ligand concentrations. Here, we have extended this work with measurements on a structurally similar ligand, S-adenosyl-L-homocysteine (SAH), which has, however, a much lower binding affinity. Using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling (HMM) analysis, we identified major conformations and determined their fractional populations and dynamics. At high Mg$^{2+}$ concentration, FRET analysis yielded four distinct conformations, which we assigned to two terminator and two antiterminator states. In the same solvent, but with SAM added at saturating concentrations, four states persisted, although their populations, lifetimes and interconversion dynamics changed. In the presence of SAH instead of SAM, HMM revealed again four well-populated states and, in addition, a weakly populated ‘hub’ state that appears to mediate conformational transitions between three of the other states. Our data show pronounced and specific effects of the SAM and SAH ligands on the RNA conformational energy landscape. Interestingly, both SAM and SAH shifted the fractional populations toward terminator folds, but only gradually, so the effect cannot explain the switching action. Instead, we propose that the noticeably accelerated dynamics of interconversion between terminator and antiterminator states upon SAM binding may be essential for control of transcription

Fluorescence lifetime imaging microscopy (FLIM) of intracellular transport by means of doubly labelled siRNA architectures

For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2’-positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by fluorescence lifetime imaging microscopy (FLIM), which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of GFP expression, which demonstrates the potential of our siRNA architectures as a tool for future development of effective siRNA

A simple route to highly active single-enzyme nanogels

Just add sugar: the synthesis of single-enzyme nanogels, a class of highly robust nanobiocatalysts, is boosted by the addition of carbohydrates. Our methodology is demonstrated with a dozen commercial proteins, spanning a large size interval and a broad domain of applications. In addition, new in-depth structural characterizations are provided.</p

Time‐resolved fluorescence anisotropy with Atto 488‐labeled phytochrome Agp1 from Agrobacterium fabrum

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time-resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto-488 was attached to each mutant, and time-resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A$_2$ of the second component between the PCM and the full-length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr-to-Pfr photoconversion induced no significant changes in the time constant t$_2$ at any position. An intermediate t$_2$ value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr