Beyond transparency: architectural application of robotically fabricated polychromatic float glass.

This research investigates robotically fabricated polychromatic float glass for architectural applications. Polychromatic glass elements usually require labor-intensive processes or are limited to film applications of secondary materials onto the glass. Previous research employs computer numerical control (CNC) based multi-channel granule deposition to manufacture polychromatic relief glass; however, it is limited in motion, channel control, and design space. To expand the design and fabrication space for the manufacture of mono-material polychromatic glass elements, this paper presents further advancements using a UR robotic arm with an advanced multi-channel dispenser, linear and curved-paths granule deposition, customized color pattern design approaches, and a computational tool for the prediction and rendering of outcomes. A large-scale demonstrator serves as a case study for upscaling. Robotic multi-channel deposition and tailored computational design tools are employed to facilitate a full-scale installation consisting of eighteen large glass panels. Novel optical properties include locally varying color, opacity, and texture filter light and view. The resulting product constructs sublime architectural experiences through light refraction, reflection, color, opacity - beyond mere transparency.

Arrestin-1 engineering facilitates complex stabilization with native rhodopsin.

Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin-arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC50) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assessed arrestin-1 mutants are still specifically associating with phosphorylated or light-activated receptor states only, while binding to the inactive ground state of the receptor is not significantly altered. Additionally, we propose a novel functionality for R171 in stabilizing the inactive arrestin-1 conformation as well as the rhodopsin-arrestin-1 complex. The achieved stabilization of the active rhodopsin-arrestin-1 complex might be of great interest for future structure determination, antibody development studies as well as drug-screening efforts targeting G protein-coupled receptors (GPCRs).

Functional map of arrestin binding to phosphorylated opsin, with and without agonist.

Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct.

Functional map of arrestin-1 at single amino acid resolution.

Arrestins function as adapter proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and additional rounds of signaling. Here we have compared binding of the GPCR rhodopsin to 403 mutants of arrestin-1 covering its complete sequence. This comprehensive and unbiased mutagenesis approach provides a functional dimension to the crystal structures of inactive, preactivated p44 and phosphopeptide-bound arrestins and will guide our understanding of arrestin-GPCR complexes. The presented functional map quantitatively connects critical interactions in the polar core and along the C tail of arrestin. A series of amino acids (Phe375, Phe377, Phe380, and Arg382) anchor the C tail in a position that blocks binding of the receptor. Interaction of phosphates in the rhodopsin C terminus with Arg29 controls a C-tail exchange mechanism in which the C tail of arrestin is released and exposes several charged amino acids (Lys14, Lys15, Arg18, Lys20, Lys110, and Lys300) for binding of the phosphorylated receptor C terminus. In addition to this arrestin phosphosensor, our data reveal several patches of amino acids in the finger (Gln69 and Asp73-Met75) and the lariat loops (L249-S252 and Y254) that can act as direct binding interfaces. A stretch of amino acids at the edge of the C domain (Trp194-Ser199, Gly337-Gly340, Thr343, and Thr345) could act as membrane anchor, binding interface for a second rhodopsin, or rearrange closer to the central loops upon complex formation. We discuss these interfaces in the context of experimentally guided docking between the crystal structures of arrestin and light-activated rhodopsin.