Seraphine V. Wegner

Dynamic Copper(I) Imaging in Mammalian Cells with a Genetically Encoded Fluorescent Copper(I) Sensor

Copper, a key cofactor for many life processes, is toxic at elevated levels, and its availability is strictly controlled inside cells. Therefore, it is a challenge to visualize copper availability in the tight copper-binding environment of the cell. We report a genetically encoded fluorescent copper(I) sensor based on the copper(I)-binding-induced conformational change of a copper-responsive transcriptional regulator, Amt1. The resulting reporter, Amt1-FRET, is ratiometric, highly sensitive (K(d) = 2.5 x 10(-18) M), and selective toward copper(I). Its measured high affinity to copper(I) confirms the extremely low copper availability in yeast since Amt1 senses the upper limit of cellular copper levels in yeast and activates copper detoxification genes. Amt1-FRET operates in the dynamic range of the cellular copper buffer in mammalian cells and can report dynamic fluctuations of the cellular copper availability within minutes of perturbation. Thus, Amt1-FRET visualizes the tightly controlled copper availability in mammalian cells.

Molecular mechanism and structure of the Saccharomyces cerevisiae iron regulator Aft2

Significance Iron is essential for eukaryotic cell survival but toxic at higher concentrations. In yeast, iron levels are tightly regulated by the transcriptional activators Aft1 and Aft2 (activators of ferrous transport), which activate iron-uptake genes when iron levels are low. We report the first crystal structure of DNA-bound Aft2 and show that Aft2 senses cellular iron levels via direct [2Fe-2S]-cluster binding, which promotes Aft2 dimerization and deactivation of the regulated genes. We further demonstrate that Aft2 acquires a [2Fe-2S] cluster from glutaredoxin-3 and Fe repressor of activation-2, two [2Fe-2S]-binding proteins with homologs in higher eukaryotes. This study unveils the molecular mechanism of the Aft family of iron-regulatory proteins and emphasizes the importance of Fe-S clusters in cellular iron sensing in eukaryotes. The paralogous iron-responsive transcription factors Aft1 and Aft2 (activators of ferrous transport) regulate iron homeostasis in Saccharomyces cerevisiae by activating expression of iron-uptake and -transport genes when intracellular iron is low. We present the previously unidentified crystal structure of Aft2 bound to DNA that reveals the mechanism of DNA recognition via specific interactions of the iron-responsive element with a Zn2+-containing WRKY-GCM1 domain in Aft2. We also show that two Aft2 monomers bind a [2Fe-2S] cluster (or Fe2+) through a Cys-Asp-Cys motif, leading to dimerization of Aft2 and decreased DNA-binding affinity. Furthermore, we demonstrate that the [2Fe-2S]-bridged heterodimer formed between glutaredoxin-3 and the BolA-like protein Fe repressor of activation-2 transfers a [2Fe-2S] cluster to Aft2 that facilitates Aft2 dimerization. Previous in vivo findings strongly support the [2Fe-2S] cluster-induced dimerization model; however, given the available evidence, Fe2+-induced Aft2 dimerization cannot be completely ruled out as an alternative Aft2 inhibition mechanism. Taken together, these data provide insight into the molecular mechanism for iron-dependent transcriptional regulation of Aft2 and highlight the key role of Fe-S clusters as cellular iron signals.

Genetically Encoded Copper(I) Reporters with Improved Response for Use in Imaging

Copper represents one of the most important biological metal ions due to its role as a catalytic cofactor in a multitude of proteins. However, an excess of copper is highly toxic. Thus, copper is heavily regulated, and copper homeostasis is controlled by many metalloregulatory proteins in various organisms. Here we report a genetically encoded copper(I) probe capable of monitoring copper fluctuations inside living cells. We insert the copper regulatory protein Ace1 into a yellow fluorescent protein, which selectively binds copper(I) and generates improved copper(I) probes.

A Genetically Encoded FRET Sensor for Intracellular Heme

Heme plays pivotal roles in various cellular processes as well as in iron homeostasis in living systems. Here, we report a genetically encoded fluorescence resonance energy transfer (FRET) sensor for selective heme imaging by employing a pair of bacterial heme transfer chaperones as the sensory components. This heme-specific probe allows spatial-temporal visualization of intracellular heme distribution within living cells.

Dual-Functionalized Nanostructured Biointerfaces by Click Chemistry

The presentation of biologically active molecules at interfaces has made it possible to investigate the responses of cells to individual molecules in their matrix at a given density and spacing. However, more sophisticated methods are needed to create model surfaces that present more than one molecule in a controlled manner in order to mimic at least partially the complexity given in natural environments. Herein, we present dual-functionalized surfaces combining quasi-hexagonally arranged gold nanoparticles with defined spacings and a newly developed PEG-alkyne coating to functionalize the glass in the intermediate space. The PEG-alkyne coating provides an inert background for cell interactions but can be modified orthogonally to the gold nanoparticles with numerous azides, including spectroscopically active molecules, peptides, and biotin at controlled densities by the copper(I)-catalyzed azide alkyne click reaction. The simultaneous presentation of cRGD on the gold nanoparticles with 100 nm spacing and synergy peptide PHSRN in the space between has a striking effect on REF cell adhesion; cells adhere, spread, and form mature focal adhesions on the dual-functionalized surfaces, whereas cells cannot adhere on either monofunctional surface. Combining these orthogonal functionalization methods creates a new platform to study precisely the crosstalk and synergy between different signaling molecules and clustering effects in ligand–receptor interactions.

Desmosine-Inspired Cross-Linkers for Hyaluronan Hydrogels

We designed bioinspired cross-linkers based on desmosine, the cross-linker in natural elastin, to prepare hydrogels with thiolated hyaluronic acid. These short, rigid cross-linkers are based on pyridinium salts (as in desmosine) and can connect two polymer backbones. Generally, the obtained semi-synthetic hydrogels are form-stable, can withstand repeated stress, have a large linear-elastic range, and show strain stiffening behavior typical for biopolymer networks. In addition, it is possible to introduce a positive charge to the core of the cross-linker without affecting the gelation efficiency, or consequently the network connectivity. However, the mechanical properties strongly depend on the charge of the cross-linker. The properties of the presented hydrogels can thus be tuned in a range important for engineering of soft tissues by controlling the cross-linking density and the charge of the cross-linker.

Selective Recognition of Americium by Peptide-Based Reagents

The separation of lanthanides from minor actinides such as americium and curium is an important step during the recycling process in the treatment of nuclear waste. However, the similar chemistry and ionic size of lanthanide and actinide ions make the separation challenging. Here, we report that a peptide-based reagent can selectively bind trivalent actinides over trivalent lanthanides by means of introducing soft-donor atoms into a peptide known as a lanthanide-binding tag (LBT). Fluorescence spectroscopy has been used to measure the dissociation constant of each metal/peptide complex. A 10-fold selectivity was obtained for Am(3+) over the similarly sized lanthanide cation, Nd(3+), when the asparagine on the fifth position of a LBT was mutated to a cysteine and further functionalized by a pyridine moiety.

Metal-binding properties of Hpn from Helicobacter pylori and implications for the therapeutic activity of bismuth

Nickel is of particular importance to Helicobacter pylori in part because it acts as a cofactor of urease, which is critical to the survival of H. pylori. In this study the nickel storage, histidine-rich protein Hpn from H. pylori was converted into a Ni2+ probe by inserting it between two fluorescence resonance energy transfer (FRET) partners, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). The resulting construct, Hpn-FRET, exhibited a change in FRET upon the binding of Ni2+. Hpn-FRET has a moderate selectivity for Ni2+; it also responds to Zn2+ and Co2+ but not to other biometals. Competition experiments between Ni2+ and other metals plus the measured Kd values for Zn2+ and Ni2+ establish the selectivity order for Hpn-FRET as Zn2+ > Ni2+ > Co2+ ≫ other biometals. Bismuth is widely used as a therapeutic agent against H. pylori, and Hpn has been suggested as one of the possible targets. The dissociation constant of Bi3+ to Hpn-FRET was measured to be 6.19 × 10−5 M. Further experiments using Hpn-FRET in E. coli indicate that Hpn-FRET responds to Bi3+ but not to Ni2+ and Zn2+ inside E. coli. The result shows that unlike Ni2+ and Zn2+, which are tightly regulated in most bacteria, available Bi3+ can reach high micromolar levels inside E. coli.

Cobalt(III)-Mediated Permanent and Stable Immobilization of Histidine-Tagged Proteins on NTA-Functionalized Surfaces.

We present the cobalt(III)-mediated interaction between polyhistidine (His)-tagged proteins and nitrilotriacetic acid (NTA)-modified surfaces as a general approach for a permanent, oriented, and specific protein immobilization. In this approach, we first form the well-established Co(2+) -mediated interaction between NTA and His-tagged proteins and subsequently oxidize the Co(2+) center in the complex to Co(3+) . Unlike conventionally used Ni(2+) - or Co(2+) -mediated immobilization, the resulting Co(3+) -mediated immobilization is resistant toward strong ligands, such as imidazole and ethylenediaminetetraacetic acid (EDTA), and washing off over time because of the high thermodynamic and kinetic stability of the Co(3+) complex. This immobilization method is compatible with a wide variety of surface coatings, including silane self-assembled monolayers (SAMs) on glass, thiol SAMs on gold surfaces, and supported lipid bilayers. Furthermore, once the cobalt center has been oxidized, it becomes inert toward reducing agents, specific and unspecific interactions, so that it can be used to orthogonally functionalize surfaces with multiple proteins. Overall, the large number of available His-tagged proteins and materials with NTA groups make the Co(3+) -mediated interaction an attractive and widely applicable platform for protein immobilization.

The spatial molecular pattern of integrin recognition sites and their immobilization to colloidal nanobeads determine α2β1 integrin-dependent platelet activation

Collagen, a strong platelet activator, is recognized by integrin α2β1 and GPVI. It induces aggregation, if added to suspended platelets, or platelet adhesion if immobilized to a surface. The recombinant non-prolylhydroxylated mini-collagen FC3 triple helix containing one α2β1 integrin binding site is a tool to specifically study how α2β1 integrin activates platelet. Whereas soluble FC3 monomers antagonistically block collagen-induced platelet activation, immobilization of several FC3 molecules to an interface or to colloidal nanobeads determines the agonistic action of FC3. Nanopatterning of FC3 reveals that intermolecular distances below 64 nm between α2β1 integrin binding sites trigger signaling through dot-like clusters of α2β1 integrin, which are visible in high resolution microscopy with dSTORM. Upon signaling, these integrin clusters increase in numbers per platelet, but retain their individual size. Immobilization of several FC3 to 100 nm-sized nanobeads identifies α2β1 integrin-triggered signaling in platelets to occur at a twentyfold slower rate than collagen, which activates platelet in a fast integrative signaling via different platelet receptors. As compared to collagen stimulation, FC3-nanobead-triggered signaling cause a significant stronger activation of the protein kinase BTK, a weak and dispensable activation of PDK1, as well as a distinct phosphorylation pattern of PDB/Akt.