Conformational dynamics of a single protein monitored for 24 hours at video rate

We use plasmon rulers to follow the conformational dynamics of a single protein for up to 24 h at a video rate. The plasmon ruler consists of two gold nanospheres connected by a single protein linker. In our experiment, we follow the dynamics of the molecular chaperone heat shock protein 90, which is known to show open and closed conformations. Our measurements confirm the previously known conformational dynamics with transition times in the second to minute time scale and reveals new dynamics on the time scale of minutes to hours. Plasmon rulers thus extend the observation bandwidth 3/4 orders of magnitude with respect to single-molecule fluorescence resonance energy transfer and enable the study of molecular dynamics with unprecedented precision

Orthogonal Light-Dependent Membrane Adhesion Induces Social Self-Sorting and Member-Specific DNA Communication in Synthetic Cell Communities

Developing orthogonal chemical communication pathways in diverse synthetic cell communities is a considerable challenge due to the increased crosstalk and interference associated with large numbers of different types of sender-receiver pairs. Herein, the authors control which sender-receiver pairs communicate in a three-membered community of synthetic cells through red and blue light illumination. Semipermeable protein-polymer-based synthetic cells (proteinosomes) with complementary membrane-attached protein adhesion communicate through single-stranded DNA oligomers and synergistically process biochemical information within a community consisting of one sender and two different receiver populations. Different pairs of red and blue light-responsive protein-protein interactions act as membrane adhesion mediators between the sender and receivers such that they self-assemble and socially self-sort into different multicellular structures under red and blue light. Consequently, distinct sender-receiver pairs come into the signaling range depending on the light illumination and are able to communicate specifically without activation of the other receiver population. Overall, this work shows how photoswitchable membrane adhesion gives rise to different self-sorting protocell patterns that mediate member-specific DNA-based communication in ternary populations of synthetic cells and provides a step towards the design of orthogonal chemical communication networks in diverse communities of synthetic cells

Reversible Social Self-Sorting of Colloidal Cell-Mimics with Blue Light Switchable Proteins

Toward the bottom-up assembly of synthetic cells from molecular building blocks, it is an ongoing challenge to assemble micrometer sized compartments that host different processes into precise multicompartmental assemblies, also called prototissues. The difficulty lies in controlling interactions between different compartments dynamically both in space and time, as these interactions determine how they organize with respect to each other and how they work together. In this study, we have been able to control the self-assembly and social self-sorting of four different types of colloids, which we use as a model for synthetic cells, into two separate families with visible light. For this purpose we used two photoswitchable protein pairs (iLID/Nano and nHagHigh/pMagHigh) that both reversibly heterodimerize upon blue light exposure and dissociate from each other in the dark. These photoswitchable proteins provide noninvasive, dynamic, and reversible remote control under biocompatible conditions over the self-assembly process with unprecedented spatial and temporal precision. In addition, each protein pair brings together specifically two different types of colloids. The orthogonality of the two protein pairs enables social self-sorting of a four component mixture into two distinct families of colloidal aggregates with controlled arrangements. These results will ultimately pave the way for the bottom-up assembly of multicompartment synthetic prototissues of a higher complexity, enabling us to control precisely and dynamically the organization of different compartments in space and time

Design of an emission ratiometric biosensor from MerR family proteins: A sensitive and selective sensor for Hg(II)

A ratiometric fluorescent biosensor for Hg2+ is constructed from the MerR protein and a duplex DNA containing a pyrene excimer

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

Cobalt Cross-Linked Redox-Responsive PEG Hydrogels: From Viscoelastic Liquids to Elastic Solids

We describe cobalt cross-linked redox-responsive 4-arm histidine-modified PEG (4A-PEG-His) hydrogels, which can be switched from self-healing viscoelastic liquids to form stable elastic solids through a simple oxidation step from Co²⁺ to Co³⁺. The dramatic change in gel properties is quantified in rheological measurements and is associated with the altered ligand exchange rate of the cross-linking cobalt ions. While Co²⁺ forms kinetically labile coordination bonds with low thermodynamic stability, Co³⁺ forms kinetically inert and highly stable coordination bonds. Unlike the Co²⁺ cross-linked hydrogels, the Co³⁺ cross-linked hydrogels do not dissolve in buffer and swell overtime, where they remain intact longer with increasing gel connectivity, increasing polymer concentration and decreasing temperature. Remarkably, these gels can even resist the strong chelator EDTA and withstand both low and high pH due to the low ligand exchange rates in the primary coordination sphere. Overall, the Co^2+/3+ redox pair provides an attractive platform to produce redox-responsive materials with big deviations in mechanical and chemical properties

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