Summary Unmodified neurons can be directly stimulated with light to produce action potentials, but such techniques have lacked localization of the delivered light energy. Here we show that gold nanoparticles can be conjugated to high-avidity ligands for a variety of cellular targets. Once bound to a neuron, these particles transduce millisecond pulses of light into heat which changes membrane capacitance, depolarizing the cell and eliciting action potentials. Compared to non-functionalized nanoparticles, ligand-conjugated nanoparticles highly resist convective washout, and enable photothermal stimulation with lower delivered energy and resulting temperature increase. Ligands targeting three different membrane proteins were tested; all showed similar activity and washout resistance. This suggests that many types of ligands can be bound to nanoparticles, preserving ligand and nanoparticle function, and that many different cell phenotypes can be targeted by appropriate choice of ligand. The findings have applications as an alternative to optogenetics, and potentially for therapies involving neuronal photostimulation.
We have re-examined the utility of native chemical ligation at −Gln/Glu-Cys− [Glx-Cys] and −Asn/Asp-Cys− [Asx-Cys] sites. Using the improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), native chemical ligation could be performed at −Gln-Cys− and Asn-Cys− sites without side reactions. After optimization, ligation at a −Glu-Cys− could also be used as a ligation site, with minimal levels of byproduct formation. However, −Asp-Cys− is not appropriate for use as a site for native chemical ligation because of formation of significant amounts of β-linked byproduct. The feasibility of native chemical ligation at −Gln-Cys− enabled a convergent total chemical synthesis of the enantiomeric forms of the ShK toxin protein molecule. The D-ShK protein molecule was ~50,000-fold less active in blocking the Kv1.3 channel than the L-ShK protein molecule. Racemic protein crystallography was used to obtain high resolution X-ray diffraction data for ShK toxin. The structure was solved by direct methods and showed significant differences from the previously reported NMR structures in some regions of the ShK protein molecule.
Covalently locking interacting proteins in situ is an attractive strategy for addressing the challenge of identifying weak and transient protein interactions, yet it is demanding to execute chemical reactions in live systems in a biocompatible, specific, and autonomous manner. Harnessing proximity-enabled reactivity of an unnatural amino acid incorporated in the bait toward a target residue of unknown proteins, here we genetically encode chemical cross-linkers (GECX) to cross-link interacting proteins spontaneously and selectively in live cells. Obviating an external trigger for reactivity and affording residue specificity, GECX enables the capture of low-affinity protein binding (affibody with Z protein), elusive enzyme-substrate interaction (ubiquitin-conjugating enzyme UBE2D3 with substrate PCNA), and endogenous proteins interacting with thioredoxin in E. coli cells, allowing for mass spectrometric identification of interacting proteins and crosslinking sites. This live cell chemistry-based approach should be valuable for investigating currently intangible protein interactions in vivo for better understanding of biology in physiological settings.
The folding of natural proteins typically relies on hydrophobic packing, metal binding, or disulfide bond formation in the protein core. Alternatively, a 3D structure can be defined by incorporating a multivalent cross-linking agent, and this approach has been successfully developed for the selection of bicyclic peptides from large random-sequence libraries. By contrast, there is no general method for the de novo computational design of multicross-linked proteins with predictable and well-defined folds, including ones not found in nature. Here we use Rosetta and Tertiary Motifs (TERMs) to design small proteins that fold around multivalent cross-linkers. The hydrophobic cross-linkers stabilize the fold by macrocyclic restraints, and they also form an integral part of a small apolar core. The designed CovCore proteins were prepared by chemical synthesis, and their structures were determined by solution NMR or X-ray crystallography. These mesosized proteins, lying between conventional proteins and small peptides, are easily accessible either through biosynthetic precursors or chemical synthesis. The unique tertiary structures and ease of synthesis of CovCore proteins indicate that they should provide versatile templates for developing inhibitors of protein-protein interactions.
Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.V oltage-gated sodium channels (Navs) play an essential role in the generation and propagation of action potentials in excitable cells (1). Eukaryotic Navs are composed of a poreforming α subunit and auxiliary β subunits. The α subunit is a large single-polypeptide chain organized in four different domains (DI-DIV), each of which has a voltage-sensing domain (VSD; S1-S4 segments) and a pore-forming domain (S5-S6 segments). Each domain has a different amino acid composition, pointing to some level of functional specialization. Site-directed fluorimetry shows that the VSDs in DI, DII, and DIII of the rat skeletal muscle voltage-gated sodium channel (Nav1.4) are activated by depolarization faster than in DIV (2). From this observation, it has been hypothesized that DI-III VSDs control the pore opening of the mammalian Nav, whereas the DIV VSD governs its fast inactivation (2-5).Although mammalian Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. The crystal structures of several prokaryotic Navs have been solved recently; however, in contrast to the mammalian Navs, they are homotetrameric, and thus structurally more closely related to the organization of voltage-gated potassium channels (Kvs) (6-9). The structure of a human L-type voltage-gated calcium channel type 1.1 (Cav1.1), which is a large single polypeptide composed of four different domains similar to mammalian Navs, has been resolved using cryoelectron microscopy (10, 11). However, functional studies have shown that gating mechanisms of mammalian Cav channels are indeed different from gating mechanisms in mammalian Navs (12). Furthermore, such structural studies only provide static snapshots of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are nee...
Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkers for prodrugs had not been developed for bacterial proteases, we used substrate phage to discover substrates for proteases found in the bacterial periplasm. Rather than focusing on a single protease, we used a periplasmic extract of E. coli to find sequences with the greatest susceptibility to the endogenous mixture of periplasmic proteases. Using a fluorescence assay, candidate sequences were evaluated to identify substrates that release native amine-containing payloads. We next designed conjugates consisting of (1) an N-terminal siderophore to facilitate uptake, (2) a protease-cleavable linker, and (3) an amine-containing antibiotic. Using this strategy, we converted daptomycinwhich by itself is active only against Gram-positive bacteriainto an antibiotic capable of targeting Gram-negative Acinetobacter species. We similarly demonstrated siderophore-facilitated delivery of oxazolidinone and macrolide antibiotics into a number of Gram-negative species. These results illustrate this platform’s utility for development of protease-activated prodrugs, including Trojan horse antibiotics.
Biocompatible chemical protein cleavage methods have been long-sought to replace enzymatic cleavages, but have yet to be realized. Here, we report the development of the SNAC-tag (Sequence-specific Nickel Assisted Cleavage) to achieve sequence-specific chemical protein cleavage under biocompatible conditions with comparable efficiency to enzymes. We demonstrate that the SNAC-tag can be inserted before both water-soluble and membrane proteins to achieve fusion protein cleavage, even when enzymatic cleavages fail.
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