Mandipropamid as a chemical inducer of proximity for in vivo applications.

Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins.

A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein-Protein Conjugates.

Bioorthogonal chemistry holds great potential to generate difficult-to-access protein-protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels-Alder cycloaddition with inverse electron demand (DAinv ). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin-based targeted cancer therapies.

Enzymatic and Site-Specific Ligation of Minimal-Size Tetrazines and Triazines to Proteins for Bioconjugation and Live-Cell Imaging.

Diels-Alder reactions with inverse electron demand (DAinv) have emerged as an indispensable tool for bioorthogonal labeling and the manipulation of biomolecules. In this context, reactions between tetrazines and strained dienophiles have received attention because of high reaction rates. Current methods for the DAinv-mediated functionalization of proteins suffer from slow reactivity, impaired stability, isomerization, or elimination of the incorporated strained dienophiles. We report here a versatile platform for the posttranslational, highly selective, and quantitative modification of proteins with stable dienes. New synthetic access to minimal size tetrazine and triazine derivatives enabled us to synthesize tailored diene substrates for the lipoic acid protein ligase A (LplA) from Escherichia coli, which we employ for the rapid, mild, and quantitative bioconjugation of proteins by DAinv. The presented method benefits from the minimal tag size for LplA recognition and can be applied to proteins from any source organism. We demonstrate its broad suitability by site-specific in vitro protein labeling and live cell labeling for fluorescence microscopy. With this work we expand the scope of DAinv bioorthogonal chemistry for site-specific protein labeling, providing additional experimental flexibility for preparing well-defined bioconjugates and addressing biological questions in complex biological environments.

An extinct north American porcupine with a South American tail.

New World porcupines (Erethizontinae) originated in South America and dispersed into North America as part of the Great American Biotic Interchange (GABI) 3-4 million years ago.1 Extant prehensile-tailed porcupines (Coendou) today live in tropical forests of Central and South America.2,3 In contrast, North American porcupines (Erethizon dorsatum) are thought to be ecologically adapted to higher-latitude temperate forests, with a larger body, shorter tail, and diet that includes bark.4,5,6,7 Limited fossils8,9,10,11,12,13 have hindered our understanding of the timing of this ecological differentiation relative to intercontinental dispersal during the GABI and expansion into temperate habitats.14,15,16,17,18 Here, we describe functionally important features of the skeleton of the extinct Erethizon poyeri, the oldest nearly complete porcupine skeleton documented from North America, found in the early Pleistocene of Florida. It differs from extant E. dorsatum in having a long, prehensile tail, grasping foot, and lacking dental specializations for bark gnawing, similar to tropical Coendou. Results from phylogenetic analysis suggest that the more arboreal characteristics found in E. poyeri are ancestral for erethizontines. Only after it expanded into temperate, Nearctic habitats did Erethizon acquire the characteristic features that it is known for today. When combined with molecular estimates of divergence times, results suggest that Erethizon was ecologically similar to a larger species of Coendou when it crossed the Isthmus of Panama by the early Pleistocene. It is likely that the range of this more tropically adapted form was limited to a continuous forested biome that extended from South America through the Gulf Coast.

Ice Age megafauna rock art in the Colombian Amazon?

Megafauna paintings have accompanied the earliest archaeological contexts across the continents, revealing a fundamental inter-relationship between early humans and megafauna during the global human expansion as unfamiliar landscapes were humanized and identities built into new territories. However, the identification of extinct megafauna from rock art is controversial. Here, we examine potential megafauna depictions in the rock art of Serranía de la Lindosa, Colombian Amazon, that includes a giant sloth, a gomphothere, a camelid, horses and three-toed ungulates with trunks. We argue that they are Ice Age rock art based on the (i) naturalistic appearance and diagnostic morphological features of the animal images, (ii) late Pleistocene archaeological dates from La Lindosa confirming the contemporaneity of humans and megafauna, (iii) recovery of ochre pigments in late Pleistocene archaeological strata, (iv) the presence of most megafauna identified in the region during the late Pleistocene as attested by archaeological and palaeontological records, and (v) widespread depiction of extinct megafauna in rock art across the Americas. Our findings contribute to the emerging picture of considerable geographical and stylistic variation of geometric and figurative rock art from early human occupations across South America. Lastly, we discuss the implications of our findings for understanding the early human history of tropical South America. This article is part of the theme issue 'Tropical forests in the deep human past'.

Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy.

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

Light-induced protein proximity by activation of gibberellic acid derivatives in living cells.

Light controlled tools are highly attractive for the modulation and manipulation of biological processes. As an external trigger light can be applied with high temporal and special control to various samples. In the recent years a number of optochemical and -genetic tools have been developed to translate the input of light into molecular changes that result in specific biological responses. Here we present a highly efficient system for light-induced protein dimerization in live cells using photocaged derivatives of the plant hormone gibberellic acid (GA3). We provide a precise protocol for a simple one-step synthesis of the photocaged CIP and its application for protein translocation in living cells.

Siderophore inspired tetra- and octadentate antenna ligands for luminescent Eu(III) and Tb(III) complexes.

Following the success of the siderophore-inspired 1,2-hydroxypyridonate (HOPO) and 2-hydroxisophthalamide (IAM) chromophores in Eu(III) and Tb(III) luminescence, we designed three new ligands bearing both chromophores. Syntheses of the octadentate ligands 3,4,3-LI-IAM-1,2-HOPO and 3,4,3-LI-1,2-HOPO-IAM, where the chromophores are attached to different positions in the (LI=linear) spermine backbone, are reported in addition to a tetradentate ligand based on 1,5-diaminopentane. The Gd(III) complexes were prepared and revealed localized triplet states typical for the IAM and HOPO chromophores. Photophysical characterization of the Eu(III) and Tb(III) complexes revealed that the chromophores need to reside at a primary amine of the spermine backbone to be efficient in lanthanide excitation. These systems help us to understand the antenna effect in siderophore inspired chromophores and could be potential targets for sensing and biological imaging applications.