Chemoselective Pd(0)-catalyzed peptide coupling in water.

[reaction: see text] Highly charged peptides ranging in length from 17 to 33 residues have been efficiently tricoupled to a trialkyne nucleus by using a Sonogashira Pd(0) coupling strategy under both acidic (pH 5.0) and basic (pH 7.5) conditions. These results demonstrate the utility of Pd(0) to construct protein-sized structures (12,000 mol wt) in aqueous milieu.

Antibacterial agents based on the cyclic D,L-alpha-peptide architecture.

The rapid emergence of bacterial infections that are resistant to many drugs underscores the need for new therapeutic agents. Here we report that six- and eight-residue cyclic d,l-alpha-peptides act preferentially on Gram-positive and/or Gram-negative bacterial membranes compared to mammalian cells, increase membrane permeability, collapse transmembrane ion potentials, and cause rapid cell death. The effectiveness of this class of materials as selective antibacterial agents is highlighted by the high efficacy observed against lethal methicillin-resistant Staphylococcus aureus infections in mice. Cyclic d,l-alpha-peptides are proteolytically stable, easy to synthesize, and can be derived from a potentially vast membrane-active sequence space. The unique abiotic structure of the cyclic peptides and their quick bactericidal action may also contribute to limit temporal acquirement of drug resistant bacteria. The low molecular weight d,l-alpha-peptides offer an attractive complement to the current arsenal of naturally derived antibiotics, and hold considerable potential in combating a variety of existing and emerging infectious diseases.

A de novo designed peptide ligase: a mechanistic investigation.

A 33-residue de novo designed peptide ligase is reported which catalyzes the template-directed condensation of suitably activated short peptides with catalytic efficiencies in excess of 10(5) ([k(cat)/K(m)]/k(uncat)). The ligase peptide, derived from natural and designed alpha-helical coiled-coil proteins, presents a surface for substrate assembly via formation of a hydrophobic core at the peptide interface. Charged residues flanking the core provide additional binding specificity through electrostatic complementarity. Addition of the template to an equimolar fragment solution results in up to 4100-fold increases in initial reaction rates. Dramatic decreases in efficiency upon mutation of charged residues or increase in ionic strength establishes the importance of electrostatic recognition to ligase efficiency. Although most of the increase in reaction efficiency is due to entropic gain from binding of substrates in close proximity, mechanistic studies with altered substrates demonstrate that the system is highly sensitive to precise ordering at the point of ligation. Taken together these results represent the first example of a peptide catalyst with designed substrate binding sites which can significantly accelerate a bimolecular reaction and support the general viability of alpha-helical protein assemblies in artificial enzyme design.

A chiroselective peptide replicator.

The origin of homochirality in living systems is often attributed to the generation of enantiomeric differences in a pool of chiral prebiotic molecules, but none of the possible physiochemical processes considered can produce the significant imbalance required if homochiral biopolymers are to result from simple coupling of suitable precursor molecules. This implies a central role either for additional processes that can selectively amplify an initially minute enantiomeric difference in the starting material, or for a nonenzymatic process by which biopolymers undergo chiroselective molecular replication. Given that molecular self-replication and the capacity for selection are necessary conditions for the emergence of life, chiroselective replication of biopolymers seems a particularly attractive process for explaining homochirality in nature. Here we report that a 32-residue peptide replicator, designed according to our earlier principles, is capable of efficiently amplifying homochiral products from a racemic mixture of peptide fragments through a chiroselective autocatalytic cycle. The chiroselective amplification process discriminates between structures possessing even single stereochemical mutations within otherwise homochiral sequences. Moreover, the system exhibits a dynamic stereochemical 'editing' function; in contrast to the previously observed error correction, it makes use of heterochiral sequences that arise through uncatalysed background reactions to catalyse the production of the homochiral product. These results support the idea that self-replicating polypeptides could have played a key role in the origin of homochirality on Earth.

Membrane partitioning of the cleavage peptide in flock house virus.

Membrane translocation of the ssRNA genome of nodaviruses has been proposed to be mediated by direct lipid-protein interactions between a postassembly autocatalytic cleavage product from the capsomere and the target membrane. We have recently shown that the 21-residue Met-->Nle variant of the N-terminal helical domain (denoted gamma(1)) of the cleavage peptide in flock house nodavirus increases membrane permeability to hydrophilic solutes and can alter both membrane structure and function, suggesting the possibility of peptide-triggered disruption of the endosomal membrane as a prelude to viral uncoating in the host cytoplasm. Elucidation of partitioning energetics would allow an assessment of the likelihood of this mechanism. We report herein complete thermodynamic characterization of the partitioning of gamma(1) to phospholipids by lipid-peptide titrations following changes in ellipticity, fluorescence signature, or calorimetric response. These experiments revealed a partitioning energy comparable to natural membrane-active peptide toxins, suggesting that the proposed mechanism may be possible. Additionally, a novel switch in the balance of partitioning forces was found: when the lipid headgroup was changed from zwitterionic to negatively charged, membrane association of the peptide became completely entropy-driven.

An animal virus-derived peptide switches membrane morphology: possible relevance to nodaviral transfection processes.

The N-terminal domain of the capsid protein cleavage product of the flock house virus (FHV) consists of 21 residues and forms an amphipathic alpha-helix, which is thought to play a crucial role in permeabilizing biological membranes for RNA translocation in the host cell. We have found that the Met --> Nle variant of this domain (denoted here as gamma1) efficiently induces the formation of the interdigitated gel phase (LbetaI) of 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayers. In situ scanning force microscopy of solid supported bilayers and fluorescence spectroscopy of peptide-treated DPPC vesicles provide evidence for the formation of acyl chain interdigitated lipid domains. It could be shown by fluorescence spectroscopy that the peptide inserts in the DPPC matrix above the main transition temperature of the lipid, while the formation of domains with decreased thickness occurs after the sample is cooled to 25 degrees C. The orientation and secondary structure of the peptide in lipid bilayers were investigated using attenuated total reflectance infrared (ATR-IR) and circular dichroism (CD) spectroscopy. These results enabled us to formulate a mechanistic model for the peptide-mediated induction of interdigitation in DPPC bilayers. Moreover, the membrane activity of gamma1 with gel phase lipids established in this study may have further implications for the infection strategy adopted by simple RNA viruses.

Stereoselection in designed three-helix bundle metalloproteins.

The stereochemical consequences of the metal-ion assisted self-assembly of parallel three-helix peptide bundles are investigated. Chiral induction in the self-assembly of systems containing extensive protein secondary structure is compared with the racemic synthesis of short metallopeptides. Isolation and characterization of the individual stereoisomers of an exchange-inert metalloprotein provide structural insights into analogous exchange-labile systems.

Emergence of symbiosis in peptide self-replication through a hypercyclic network.

Symbiosis is an association between different organisms that leads to a reciprocal enhancement of their ability to survive. Similar mutually beneficial relationships can operate at the molecular level in the form of a hypercycle, a collective of two or more self-replicating species interlinked through a cyclic catalytic network. The superposition of cross-catalysis onto autocatalytic replication integrates the members of the hypercycle into a single system that reproduces through a second-order (or higher) form of nonlinear autocatalysis. The hypercycle population as a whole is therefore able to compete more efficiently for existing resources than any one member on its own. In addition, the effects of beneficial mutations of any one member are spread over the entire population. The formation of hypercycles has been suggested as an important step in the transition from inanimate to living chemistry, and a large number of hypercycles are expected to be embedded within the complex networks of living systems. But only one naturally occurring hypercycle has been well documented, while two autocatalytic chemical systems may contain vestiges of hypercyclic organization. Here we report a chemical system that constitutes a clear example of a minimal hypercyclic network, in which two otherwise competitive self-replicating peptides symbiotically catalyse each others' production.

A porous silicon-based optical interferometric biosensor.

A biosensor has been developed based on induced wavelength shifts in the Fabry-Perot fringes in the visible-light reflection spectrum of appropriately derivatized thin films of porous silicon semiconductors. Binding of molecules induced changes in the refractive index of the porous silicon. The validity and sensitivity of the system are demonstrated for small organic molecules (biotin and digoxigenin), 16-nucleotide DNA oligomers, and proteins (streptavidin and antibodies) at pico- and femtomolar analyte concentrations. The sensor is also highly effective for detecting single and multilayered molecular assemblies.

A synthetic peptide ligase.

The preparation of synthetic molecules showing the remarkable efficiencies characteristic of natural biopolymer catalysts remains a formidable challenge for chemical biology. Although significant advances have been made in the understanding of protein structure and function, the de novo construction of such systems remains elusive. Re-engineered natural enzymes and catalytic antibodies, possessing tailored binding pockets with appropriately positioned functional groups, have been successful in catalysing a number of chemical transformations, sometimes with impressive efficiencies. But efforts to produce wholly synthetic catalytic peptides have typically resulted in compounds with questionable structural stability, let alone reactivity. Here we describe a 33-residue synthetic peptide, based on the coiled-coil structural motif, which efficiently catalyses the condensation of two shorter peptide fragments with high sequence- and diastereoselectivity. Depending on the substrates used, we observe rate enhancements of tenfold to 4,100-fold over the background, with catalytic efficiencies in excess of 10(4). These results augur well for the rational design of functional peptides.

Autocatalytic networks: the transition from molecular self-replication to molecular ecosystems.

The transition from inanimate to animate chemistry is thought to involve self-organised networks of molecular species whose collective emergent property gives rise to the overall characteristics of living systems. In the past, simple autocatalytic networks have been constructed that display basic forms of cooperative behaviour. These include reciprocal catalysis, autocratic, and hypercyclic networks. The design and emergent properties of these novel molecular networks are reviewed here.

A self-replicating peptide.

The production of amino acids and their condensation to polypeptides under plausibly prebiotic conditions have long been known. But despite the central importance of molecular self-replication in the origin of life, the feasibility of peptide self-replication has not been established experimentally. Here we report an example of a self-replicating peptide. We show that a 32-residue alpha-helical peptide based on the leucine-zipper domain of the yeast transcription factor GCN4 can act autocatalytically in templating its own synthesis by accelerating the thioester-promoted amide-bond condensation of 15- and 17-residue fragments in neutral, dilute aqueous solutions. The self-replication process displays parabolic growth pattern with the initial rates of product formation correlating with the square-foot of initial template concentration.

Artificial transmembrane ion channels from self-assembling peptide nanotubes.

Naturally occurring membrane channels and pores are formed from a large family of diverse proteins, peptides and organic secondary metabolites whose vital biological functions include control of ion flow, signal transduction, molecular transport and production of cellular toxins. But despite the availability of a large amount of biochemical information about these molecules, the design and synthesis of artificial systems that can mimic the biological function of natural compounds remains a formidable task. Here we present a simple strategy for the design of artificial membrane ion channels based on a self-assembled cylindrical beta-sheet peptide architecture. Our systems--essentially stacks of peptide rings--display good channel-mediated ion-transport activity with rates exceeding 10(7) ions s-1, rivalling the performance of many naturally occurring counterparts. Such molecular assemblies should find use in the design of novel cytotoxic agents, membrane transport vehicles and drug-delivery systems.

Self-assembling organic nanotubes based on a cyclic peptide architecture.

Hollow tubular structures of molecular dimensions may offer a variety of applications in chemistry, biochemistry and materials science. Concentric carbon nanotubes have attracted a great deal of attention, while the three-dimensional tubular pore structures of molecular sieves have long been exploited industrially. Nanoscale tubes based on organic materials have also been reported previously. Here we report the design, synthesis and characterization of a new class of organic nanotubes based on rationally designed cyclic polypeptides. When protonated, these compounds crystallize into tubular structures hundreds of nanometres long, with internal diameters of 7-8 A. Support for the proposed tubular structures is provided by electron microscopy, electron diffraction, Fourier-transform infrared spectroscopy and molecular modelling. These tubes are open-ended, with uniform shape and internal diameter. We anticipate that they may have possible applications in inclusion chemistry, catalysis, molecular electronics and molecular separation technology.