H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 affects MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an 'electrostatic zipper' between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their 'half-open' conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.
Magnetic-activated cell sorting (MACS) is an affinity-based technique used to separate cells according to the presence of specific markers. Current MACS systems generally require an antigen to be expressed at the cell surface; these antigen-presenting cells subsequently interact with antibody-labeled magnetic particles, facilitating separation. Here, we present an alternative MACS method based on coiled-coil peptide interactions. We demonstrate that HeLa, CHO, and NIH3T3 cells can either incorporate a lipid-modified coiled-coil-forming peptide into their membrane, or that the cells can be transfected with a plasmid containing a gene encoding a coiled-coil-forming peptide. Iron oxide particles are functionalized with the complementary peptide and, upon incubation with the cells, labeled cells are facilely separated from nonlabeled populations. In addition, the resulting cells and particles can be treated with trypsin to facilitate detachment of the cells from the particles. Therefore, our new MACS method promotes efficient cell sorting of different cell lines, without the need for antigen presentation, and enables simple detachment of the magnetic particles from cells after the sorting process. Such a system can be applied to rapidly developing, sensitive research areas, such as the separation of genetically modified cells from their unmodified counterparts.
Abstract Recent attempts to mimic enzyme catalysis using simple, short peptides have been successful in enhancing various reactions, but the on‐demand, temporal or spatial regulation of such processes by external triggers remains a great challenge. Light irradiation is an ideal trigger for regulating molecular functionality, since it can be precisely manipulated in time and space, and because most reaction mediums do not react to light. We herein report the selection of a photo‐switchable amphiphilic peptide catalyst from a small library of isomeric peptides, each containing an azobenzene‐based light responsive group and a catalytic histidine residue. In its native fibrillar form, the selected peptide is efficiently and enantio‐selectively active for ester hydrolysis, but after irradiation by UV light inducing trans ‐to‐ cis azobenzene isomerization, the fibrils disassemble to amorphous aggregates that are much less catalytically active. Significantly, this esterase‐like activity can be manipulated multiple times, as the fibrillar peptide assembly is reversibly reduced and restored upon alternate irradiation by UV and visible light, respectively. We propose that this research may shine light on the origin of complex functions in early chemical evolution. Furthermore, it paves the way to regulate additional functions for peptide nanotechnology, such as replication, charge transfer, and delivery.
An ideal nanomedicine system improves the therapeutic efficacy of drugs. However, most nanomedicines enter cells via endosomal/lysosomal pathways and only a small fraction of the cargo enters the cytosol inducing therapeutic effects. To circumvent this inefficiency, alternative approaches are desired. Inspired by fusion machinery found in nature, synthetic lipidated peptide pair E4/K4 is used to induce membrane fusion previously. Peptide K4 interacts specifically with E4, and it has a lipid membrane affinity and resulting in membrane remodeling. To design efficient fusogens with multiple interactions, dimeric K4 variants are synthesized to improve fusion with E4-modified liposomes and cells. The secondary structure and self-assembly of dimers are studied; the parallel PK4 dimer forms temperature-dependent higher-order assemblies, while linear K4 dimers form tetramer-like homodimers. The structures and membrane interactions of PK4 are supported by molecular dynamics simulations. Upon addition of E4, PK4 induced the strongest coiled-coil interaction resulting in a higher liposomal delivery compared to linear dimers and monomer. Using a wide spectrum of endocytosis inhibitors, membrane fusion is found to be the main cellular uptake pathway. Doxorubicin delivery results in efficient cellular uptake and concomitant antitumor efficacy. These findings aid the development of efficient delivery systems of drugs into cells using liposome-cell fusion strategies.
A model system for membrane fusion, inspired by SNARE proteins and based on two complementary lipopeptides CPnE4 and CPnK4, has been recently developed. It consists of cholesterol (C), a poly(ethylene glycol) linker (Pn) and either a cationic peptide K4 (KIAALKE)4 or an anionic peptide E4 (EIAALEK)4. In this paper, fluorescence spectroscopy is used to decipher distinct but complementary roles of these lipopeptides during early stages of membrane fusion. Molecular evidence is provided that different distances of E4 in CPnE4 and K4 in CPnK4 from the bilayer represent an important mechanism, which enables fusion. Whereas E4 is exposed to the bulk and solely promotes membrane binding of CPnK4, K4 loops back to the lipid-water interface where it fulfills two distinct roles: it initiates bilayer contact by binding to CPnE4 containing bilayers; and it initiates fusion by modulating the bilayer properties. The interaction between CPnE4 and CPnK4 is severely down-regulated by binding of K4 to the bilayer and possible only if the lipopeptides approach each other as constituents of different bilayers. When the complementary lipopeptides are localized in the same bilayer, hetero-coiling is disabled. These data provide crucial insights as to how fusion is initiated and highlight the importance of both peptides in this process.
A parallel heterodimeric coiled coil can be mutated to an antiparallel tetrameric species by reversing the sequences of one of the peptides. This tetramer is capable of facilitating fast, efficient, membrane fusion of liposomes.
Abstract The de novo design of systems comprising peptides and proteins as building blocks presents exciting new challenges in the areas of self‐assembly and supramolecular chemistry and biochemistry. A key issue is that the rules relating protein sequence‐to‐structure are not fully elucidated. However, through the use of rational, “bottom‐up” approaches, systems can be constructed by the careful design and programming of peptidic tectons that use existing sequence‐to‐structure relationships and test new ones. Indeed, there has been considerable success in the design and development of effectively infinite, fibrous systems constructed from α‐helical, β‐structured or collagen‐based tectons. Some designs are now well enough developed that they can be readily functionalized, leading to potential applications in bionanotechnology, synthetic biology, and tissue engineering. The design of discrete assemblies using similar approaches, however, is proving more challenging, with precise control of assembly being particularly difficult. As a result, there are fewer examples of such systems being reduced to practice. This chapter describes design rules and approaches exemplified with fibrous and discrete systems, as well as discussing routes to the functionalization of such assemblies and their potential applications.
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