Plastid-localized xanthorhodopsin increases diatom biomass and ecosystem productivity in iron-limited surface oceans.

Microbial rhodopsins are photoreceptor proteins that convert light into biological signals or energy. Proteins of the xanthorhodopsin family are common in eukaryotic photosynthetic plankton including diatoms. However, their biological role in these organisms remains elusive. Here we report on a xanthorhodopsin variant (FcR1) isolated from the polar diatom Fragilariopsis cylindrus. Applying a combination of biophysical, biochemical and reverse genetics approaches, we demonstrate that FcR1 is a plastid-localized proton pump which binds the chromophore retinal and is activated by green light. Enhanced growth of a Thalassiora pseudonana gain-of-function mutant expressing FcR1 under iron limitation shows that the xanthorhodopsin proton pump supports growth when chlorophyll-based photosynthesis is iron-limited. The abundance of xanthorhodopsin transcripts in natural diatom communities of the surface oceans is anticorrelated with the availability of dissolved iron. Thus, we propose that these proton pumps convey a fitness advantage in regions where phytoplankton growth is limited by the availability of dissolved iron.

Design principles for engineering light-controlled antibodies.

Engineered antibodies are essential tools for research and advanced pharmacy. In the development of therapeutics, antibodies are excellent candidates as they offer both target recognition and modulation. Thanks to the latest advances in biotechnology, light-activated antibody fragments can be constructed to control spontaneous antigen interaction with high spatiotemporal precision. To implement conditional antigen binding, several optogenetic and optochemical engineering concepts have recently been developed. Here, we highlight the various strategies and discuss the features of opto-conditional antibodies. Each concept offers intrinsic advantages beneficial to different applications. In summary, the novel design approaches constitute a complementary toolset to promote current and upcoming antibody technologies with ultimate precision.

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies.

Genetic code expansion is a versatile method for in situ synthesis of modified proteins. During mRNA translation, amber stop codons are suppressed to site-specifically incorporate non-canonical amino acids. Thus, nanobodies can be equipped with photocaged amino acids to control target binding on demand. The efficiency of amber suppression and protein synthesis can vary with unpredictable background expression, and the reasons are hardly understood. Here, we identified a substantial limitation that prevented synthesis of nanobodies with N-terminal modifications for light control. After systematic analyses, we hypothesized that nanobody synthesis was severely affected by ribosomal inaccuracy during the early phases of translation. To circumvent a background-causing read-through of a premature stop codon, we designed a new suppression concept based on ribosomal skipping. As an example, we generated intrabodies with photoactivated target binding in mammalian cells. The findings provide valuable insights into the genetic code expansion and describe a versatile synthesis route for the generation of modified nanobodies that opens up new perspectives for efficient site-specific integration of chemical tools. In the area of photopharmacology, our flexible intrabody concept builds an ideal platform to modulate target protein function and interaction.

Light-guided intrabodies for on-demand in situ target recognition in human cells.

Due to their high stability and specificity in living cells, fluorescently labeled nanobodies are perfect probes for visualizing intracellular targets at an endogenous level. However, intrabodies bind unrestrainedly and hence may interfere with the target protein function. Here, we report a strategy to prevent premature binding through the development of photo-conditional intrabodies. Using genetic code expansion, we introduce photocaged amino acids within the nanobody-binding interface, which, after photo-activation, show instantaneous binding of target proteins with high spatiotemporal precision inside living cells. Due to the highly stable binding, light-guided intrabodies offer a versatile platform for downstream imaging and regulation of target proteins.

Ultrafast in-gel detection by fluorescent super-chelator probes with HisQuick-PAGE.

Polyacrylamide gel electrophoresis (PAGE) and immunoblotting (Western blotting) are the most common methods in life science. In conjunction with these methods, the polyhistidine-tag has proven to be a superb fusion tag for protein purification as well as specific protein detection by immunoblotting, which led to a vast amount of commercially available antibodies. Nevertheless, antibody batch-to-batch variations and nonspecific binding complicate the laborious procedure. The interaction principle applied for His-tagged protein purification by metal-affinity chromatography using N-nitrilotriacetic acid (NTA) was employed to develop small high-affinity lock-and-key molecules coupled to a fluorophore. These multivalent NTA probes allow specific detection of His-tagged proteins by fluorescence. Here, we report on HisQuick-PAGE as a fast and versatile immunoblot alternative, using such high-affinity fluorescent super-chelator probes. The procedure allows direct, fast, and ultra-sensitive in-gel detection and analysis of soluble proteins as well as intact membrane protein complexes and macromolecular ribonucleoprotein particles.

The Scaffold Design of Trivalent Chelator Heads Dictates Affinity and Stability for Labeling His-tagged Proteins in vitro and in Cells.

Small chemical/biological interaction pairs are at the forefront in tracing protein function and interaction at high signal-to-background ratios in cellular pathways. However, the optimal design of scaffold, linker, and chelator head still deserve systematic investigation to achieve the highest affinity and kinetic stability for in vitro and especially cellular applications. We report on a library of N-nitrilotriacetic acid (NTA)-based multivalent chelator heads (MCHs) built on linear, cyclic, and dendritic scaffolds and compare these with regard to their binding affinity and stability for the labeling of cellular His-tagged proteins. Furthermore, we describe a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we outline fundamental differences between the MCH scaffolds and define a cyclic trisNTA chelator that displays the highest affinity and kinetic stability of all reported reversible, low-molecular-weight interaction pairs.

Super-Chelators for Advanced Protein Labeling in Living Cells.

Live-cell labeling, super-resolution microscopy, single-molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N-nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA-based small interaction pairs described so far. Coupled to bright organic fluorophores with fine-tuned photophysical properties, the super-chelator probes were delivered into human cells by chemically gated nanopores. These super-chelators permit kinetic profiling, multiplexed labeling of His6 - and His12 -tagged proteins as well as single-molecule-based super-resolution imaging.