Caveolae disassemble upon membrane lesioning and foster cell survival.

Repair of lesions in the plasma membrane is key to sustaining cellular homeostasis. Cells maintain cytoplasmic as well as membrane-bound stores of repair proteins that can rapidly precipitate at the site of membrane lesions. However, little is known about the origins of lipids and proteins for resealing and repair of the plasma membrane. Here we study the dynamics of caveolar proteins after laser-induced lesioning of plasma membranes of mammalian C2C12 tissue culture cells and muscle cells of intact zebrafish embryos. Single-molecule diffusivity measurements indicate that caveolar clusters break up into smaller entities after wounding. Unlike Annexins and Dysferlin, caveolar proteins do not accumulate at the lesion patch. In caveolae-depleted cavin1a knockout zebrafish embryos, lesion patch formation is impaired, and injured cells show reduced survival. Our data suggest that caveolae disassembly releases surplus plasma membrane near the lesion to facilitate membrane repair after initial patch formation for emergency sealing.

Stimulated emission double depletion nanoscopy with background correction at the single-pixel level.

Fluorescence microscopy images are inevitably tainted by background contributions including emission from out-of-focus planes, scattered light, and detector noise. In stimulated emission depletion (STED) nanoscopy, an additional, method-specific background arises from incomplete depletion and re-excitation by the depletion beam. Various approaches have been proposed to remove the background from a STED image, some of which rely on the acquisition of a separate background image that is subtracted from the STED image with a weighting factor. Using stimulated emission double depletion (STEDD) nanoscopy, we observed that the weighting factor varies locally in densely labeled samples, so that background removal with a single (global) weighting factor generates local image artifacts due to incorrect background subtraction. Here we present an algorithm that computes the optimal weighting factor at the single-pixel level, yielding a difference image with excellent suppression of low-frequency background.

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.

Homodimerization Counteracts the Detrimental Effect of Nitrogenous Heme Ligands on the Enzymatic Activity of Acanthamoeba castellanii CYP51.

Acanthamoeba castellanii is a free-living amoeba that can cause severe eye and brain infections in humans. At present, there is no uniformly effective treatment for any of these infections. However, sterol 14α-demethylases (CYP51s), heme-containing cytochrome P450 enzymes, are known to be validated drug targets in pathogenic fungi and protozoa. The catalytically active P450 form of CYP51 from A. castellanii (AcCYP51) is stabilized against conversion to the inactive P420 form by dimerization. In contrast, Naegleria fowleri CYP51 (NfCYP51) is monomeric in its active P450 and inactive P420 forms. For these two CYP51 enzymes, we have investigated the interplay between the enzyme activity and oligomerization state using steady-state and time-resolved UV-visible absorption spectroscopy. In both enzymes, the P450 → P420 transition is favored under reducing conditions. The transition is accelerated at higher pH, which excludes a protonated thiol as the proximal ligand in P420. Displacement of the proximal thiolate ligand is also promoted by adding exogenous nitrogenous ligands (N-ligands) such as imidazole, isavuconazole, and clotrimazole that bind at the opposite, distal heme side. In AcCYP51, the P450 → P420 transition is faster in the monomer than in the dimer, indicating that the dimeric assembly is critical for stabilizing thiolate coordination to the heme and thus for sustaining AcCYP51 activity. The spectroscopic experiments were complemented with size-exclusion chromatography and X-ray crystallography studies. Collectively, our results indicate that effective inactivation of the AcCYP51 function by azole drugs is due to synergistic interference with AcCYP51 dimerization and promoting irreversible displacement of the proximal heme-thiolate ligand.

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.

A chemical probe for BAG1 targets androgen receptor-positive prostate cancer through oxidative stress signaling pathway.

BAG1 is a family of polypeptides with a conserved C-terminal BAG domain that functions as a nucleotide exchange factor for the molecular chaperone HSP70. BAG1 proteins also control several signaling processes including proteostasis, apoptosis, and transcription. The largest isoform, BAG1L, controls the activity of the androgen receptor (AR) and is upregulated in prostate cancer. Here, we show that BAG1L regulates AR dynamics in the nucleus and its ablation attenuates AR target gene expression especially those involved in oxidative stress and metabolism. We show that a small molecule, A4B17, that targets the BAG domain downregulates AR target genes similar to a complete BAG1L knockout and upregulates the expression of oxidative stress-induced genes involved in cell death. Furthermore, A4B17 outperformed the clinically approved antagonist enzalutamide in inhibiting cell proliferation and prostate tumor development in a mouse xenograft model. BAG1 inhibitors therefore offer unique opportunities for antagonizing AR action and prostate cancer growth.

Protein adsorption onto nanomaterials engineered for theranostic applications.

The key role of biomolecule adsorption onto engineered nanomaterials for therapeutic and diagnostic purposes has been well recognized by the nanobiotechnology community, and our mechanistic understanding of nano-bio interactions has greatly advanced over the past decades. Attention has recently shifted to gaining active control of nano-bio interactions, so as to enhance the efficacy of nanomaterials in biomedical applications. In this review, we summarize progress in this field and outline directions for future development. First, we briefly review fundamental knowledge about the intricate interactions between proteins and nanomaterials, as unraveled by a large number of mechanistic studies. Then, we give a systematic overview of the ways that protein-nanomaterial interactions have been exploited in biomedical applications, including the control of protein adsorption for enhancing the targeting efficiency of nanomedicines, the design of specific protein adsorption layers on the surfaces of nanomaterials for use as drug carriers, and the development of novel nanoparticle array-based sensors based on nano-bio interactions. We will focus on particularly relevant and recent examples within these areas. Finally, we conclude this topical review with an outlook on future developments in this fascinating research field.

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 Mg2+ 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 Mg2+ 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.

Fluorescent proteins of the EosFP clade: intriguing marker tools with multiple photoactivation modes for advanced microscopy.

Optical fluorescence microscopy has taken center stage in the exploration of biological structure and dynamics, especially on live specimens, and super-resolution imaging methods continue to deliver exciting new insights into the molecular foundations of life. Progress in the field, however, crucially hinges on advances in fluorescent marker technology. Among these, fluorescent proteins (FPs) of the GFP family are advantageous because they are genetically encodable, so that live cells, tissues or organisms can produce these markers all by themselves. A subclass of them, photoactivatable FPs, allow for control of their fluorescence emission by light irradiation, enabling pulse-chase imaging and super-resolution microscopy. In this review, we discuss FP variants of the EosFP clade that have been optimized by amino acid sequence modification to serve as markers for various imaging techniques. In general, two different modes of photoactivation are found, reversible photoswitching between a fluorescent and a nonfluorescent state and irreversible green-to red photoconversion. First, we describe their basic structural and optical properties. We then summarize recent research aimed at elucidating the photochemical processes underlying photoactivation. Finally, we briefly introduce various advanced imaging methods facilitated by specific EosFP variants, and show some exciting sample applications.

Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics.

Overcoming limitations of previous fluorescent light-up RNA aptamers for super-resolution imaging, we present RhoBAST, an aptamer that binds a fluorogenic rhodamine dye with fast association and dissociation kinetics. Its intermittent fluorescence emission enables single-molecule localization microscopy with a resolution not limited by photobleaching. We use RhoBAST to image subcellular structures of RNA in live and fixed cells with about 10-nm localization precision and a high signal-to-noise ratio.

Brownian motion-based nanoparticle sizing-A powerful approach for in situ analysis of nanoparticle-protein interactions.

A key hurdle toward effective application of nanoparticles (NPs) in biomedicine is still the incomplete understanding of the biomolecular adsorption layer, the so-called protein corona, which inevitably forms around NPs when they are immersed in a biofluid. NP sizing techniques via the analysis of Brownian motions offer a powerful way to measure the thickness of the protein corona in situ. Here, the fundamentals of three techniques, dynamic light scattering, fluorescence correlation spectroscopy, and nanoparticle tracking analysis are briefly summarized. Then, experimental procedures for the determination of binding curves are presented in a tutorial fashion. Nanoparticle sizing experiments are illustrated with a selection of recent results on the interactions of transferrin with hydrophilic and hydrophobic polystyrene nanoparticles, and key insights gained from this work are discussed.

Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy.

Development and homeostasis of multicellular organisms is largely controlled by complex cell-cell signaling networks that rely on specific binding of secreted ligands to cell surface receptors. The Wnt signaling network, as an example, involves multiple ligands and receptors to elicit specific cellular responses. To understand the mechanisms of such a network, ligand-receptor interactions should be characterized quantitatively, ideally in live cells or tissues. Such measurements are possible using fluorescence microscopy yet challenging due to sample movement, low signal-to-background ratio and photobleaching. Here, we present a robust approach based on fluorescence correlation spectroscopy with ultra-high speed axial line scanning, yielding precise equilibrium dissociation coefficients of interactions in the Wnt signaling pathway. Using CRISPR/Cas9 editing to endogenously tag receptors with fluorescent proteins, we demonstrate that the method delivers precise results even with low, near-native amounts of receptors.

Formation of a Monolayer Protein Corona around Polystyrene Nanoparticles and Implications for Nanoparticle Agglomeration.

Nanoparticle (NP) interactions with cells and organisms are mediated by a biomolecular adsorption layer, the so-called "protein corona." An in-depth understanding of the corona is a prerequisite to successful and safe application of NPs in biology and medicine. In this work, earlier in situ investigations on small NPs are extended to large polystyrene (PS) NPs of up to 100 nm diameter, using human transferrin (Tf) and human serum albumin (HSA) as model proteins. Direct NP sizing experiments reveal a reversibly bound monolayer protein shell (under saturating conditions) on hydrophilic, carboxyl-functionalized (PS-COOH) NPs, as was earlier observed for much smaller NPs. In contrast, protein binding on hydrophobic, sulfated (PS-OSO3 H) NPs in solvent of low ionic strength is completely irreversible; nevertheless, the thickness of the observed protein corona again corresponds to a protein monolayer. Under conditions of reduced charge repulsion (higher ionic strength), the NPs are colloidally unstable and form large clusters below a certain protein-NP stoichiometric ratio, indicating that the adsorbed proteins induce NP agglomeration. This comprehensive characterization of the persistent protein corona on PS-OSO3 H NPs by nanoparticle sizing and quantitative fluorescence microscopy/nanoscopy reveals mechanistic aspects of molecular interactions occurring during exposure of NPs to biofluids.

Pulsed interleaved excitation-based line-scanning spatial correlation spectroscopy (PIE-lsSCS).

We report pulsed interleaved excitation (PIE) based line-scanning spatial correlation spectroscopy (PIE-lsSCS), a quantitative fluorescence microscopy method for the study of dynamics in free-standing lipid bilayer membranes. Using a confocal microscope, we scan multiple lines perpendicularly through the membrane, each one laterally displaced from the previous one by several ten nanometers. Scanning through the membrane enables us to eliminate intensity fluctuations due to membrane displacements with respect to the observation volume. The diffusion of fluorescent molecules within the membrane is quantified by spatial correlation analysis, based on the fixed lag times between successive line scans. PIE affords dual-color excitation within a single line scan and avoids channel crosstalk. PIE-lsSCS data are acquired from a larger membrane region so that sampling is more efficient. Moreover, the local photon flux is reduced compared with single-point experiments, resulting in a smaller fraction of photobleached molecules for identical exposure times. This is helpful for precise measurements on live cells and tissues. We have evaluated the method with experiments on fluorescently labeled giant unilamellar vesicles (GUVs) and membrane-stained live cells.

Kinetic Study of Ligand Binding and Conformational Changes in Inducible Nitric Oxide Synthase.

Nitric oxide synthases (NOSs) are heme enzymes that generate highly reactive nitric oxide from l-arginine (l-Arg) in a complex mechanism that is still only partially understood. We have studied carbon monoxide (CO) binding to the oxygenase domain of murine inducible NOS (iNOS) by using flash photolysis. The P420 and P450 forms of the enzyme, assigned to a protonated and unprotonated proximal cysteine, through which the heme is anchored to the protein, show markedly different CO rebinding properties. The data suggest that P420 has a widely open distal pocket that admits water. CO rebinding to the P450 form strongly depends on the presence of the substrate l-Arg, the intermediate Nω-hydroxy-l-arginine, and the cofactor tetrahydrobiopterin. After adding these small molecules to the enzyme solution, the CO kinetics change slowly over the hours. This process can be described as a relaxation from a fast rebinding, metastable species to a slowly rebinding, thermodynamically stable species, which we associate with the enzymatically active form. Our results allow us to determine kinetic parameters of l-Arg binding to the ferrous deoxy iNOS protein for the first time and also provide clues regarding the nature of structural differences between the two interconverting species.

"siRNA traffic lights": arabino-configured 2'-anchors for fluorescent dyes are key for dual color readout in cell imaging.

Two fluorescent dyes covalently attached in diagonal interstrand orientation to siRNA undergo energy transfer and thereby enable a dual color fluorescence readout (red/green) for hybridization. Three different structural variations were carried out and compared by their optical properties, including (i) the base surrogate approach with an acyclic linker as a substitute of the 2-deoxyriboside between the phosphodiester bridges, (ii) the 2'-modification of conventional ribofuranosides and (iii) the arabino-configured 2'-modification. The double stranded siRNA with the latter type of modification delivered the best energy transfer efficiency, which was explained by molecular dynamics simulations that showed that the two dyes are more flexible at the arabino-configured sugars compared to the completely stacked situation at the ribo-configured ones. Single molecule fluorescence lifetime measurements indicate their application in fluorescence cell imaging, which reveals a red/green fluorescence contrast in particular for the arabino-configured 2'-modification by the two dyes, which is key for tracking of siRNA transport into HeLa cells.

The protein corona on nanoparticles as viewed from a nanoparticle-sizing perspective.

Most surfaces of engineered nanoparticles (NPs) are reactive toward biomolecules. Therefore, whenever NPs become immersed in biological fluids, proteins and other biomolecules bind to the NP surface, forming an adsorption layer (biomolecular corona) that modifies the NPs' physicochemical properties and subsequent interactions with living systems. Its exploration is a formidable endeavor owing to the enormous diversity of engineered NPs in terms of their physicochemical properties and the vast number of biomolecules available in biofluids that may bind to NPs with widely different strengths. Significant progress has been made in our understanding of the biomolecular corona, but even very basic issues are still controversially debated. In fact, there are divergent views of its microscopic structure and dynamics, even on physical properties, such as its thickness. As an example, there is no agreement on whether proteins form monolayers or multilayers upon adsorption. In our quantitative studies of NP-protein interactions by in situ fluorescence correlation spectroscopy (FCS) with highly defined model NPs and important serum proteins, we have universally observed protein monolayer formation around NPs under saturation or even oversaturation conditions. Here, we critically discuss biomolecular corona characterization using FCS and dynamic light scattering and identify challenges and future opportunities. Further careful, quantitative experiments are needed to elucidate the mechanisms of biomolecular corona formation and to characterize its structure. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Substrate Binding Primes Human Tryptophan 2,3-Dioxygenase for Ligand Binding.

The human heme enzyme tryptophan 2,3-dioxygenase (hTDO) catalyzes the insertion of dioxygen into its cognate substrate, l-tryptophan (l-Trp). Its active site structure is highly dynamic, and the mechanism of enzyme-substrate-ligand complex formation and the ensuing enzymatic reaction is not yet understood. Here we have studied complex formation in hTDO by using time-resolved optical and infrared spectroscopy with carbon monoxide (CO) as a ligand. We have observed that both substrate-free and substrate-bound hTDO coexist in two discrete conformations with greatly different ligand binding rates. In the fast rebinding hTDO conformation, there is facile ligand access to the heme iron, but it is greatly hindered in the slowly rebinding conformation. Spectroscopic evidence implicates active site solvation as playing a crucial role for the observed kinetic differences. Substrate binding shifts the conformational equilibrium markedly toward the fast species and thus primes the active site for subsequent ligand binding, ensuring that formation of the ternary complex occurs predominantly by first binding l-Trp and then the ligand. Consequently, the efficiency of catalysis is enhanced because O2 binding prior to substrate binding, resulting in nonproductive oxidation of the heme iron, is greatly suppressed.

Rational Engineering of Photoconvertible Fluorescent Proteins for Dual-Color Fluorescence Nanoscopy Enabled by a Triplet-State Mechanism of Primed Conversion.

Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create "pr"- (for primed convertible) variants of most known green-to-red pcFPs.

Substrate binding in human indoleamine 2,3-dioxygenase 1: A spectroscopic analysis.

Human indoleamine 2,3-dioxygenase (hIDO1) is a heme enzyme that catalyzes the oxidative cleavage of the L-tryptophan indole ring. As increased levels of hIDO1 expression in tumor cells correlate with a poor prognosis for surviving several cancer types, hIDO1 has become an appealing drug target for cancer therapy. However, detailed structural knowledge of the catalytically active complex is necessary to eb able to design de novo inhibitors selective for hIDO1. Here we have applied Fourier transform infrared (FTIR) and nanosecond time-resolved optical spectroscopy to hIDO1 variants with modified heme pocket structures to identify important amino acid residues that stabilize the substrate in the active site. A cluster of small side chain residues at positions 260-265 ensures structural flexibility of the binding site. Thr379 and Arg231 are key residues acting in concert to bind the substrate. Thr379 is the final residue of a disordered loop; the neighboring Gly380, however, is still visible in the X-ray structure of the substrate-free protein, 20Å away from the heme iron. Therefore, large-scale conformational changes are necessary to bring Thr379 close to the substrate. The use of substrate analogs further reveals that an indole-like side chain with two aromatic rings and L-stereoisomery at the Cα are required for high affinity binding.