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In living organisms most chemical reactions take place within the confines of lipid-membrane bound compartments, while confinement within the bounds of a lipid membrane is thought to be a key step in abiogenesis. In previous work we demonstrated that confinement in the aqueous cavity of a lipid vesicle affords protection against hydrolysis, a phenomenon that we term here confinement effect (C e) and that we attributed to the interaction with the lipid membrane. Here, we show that both the size and the shape of the cavity of the vesicle modulate the C e. We link this observation to the packing of the lipid following changes in membrane curvature, and formulate a mathematical model that relates the C e to the radius of a spherical vesicle and the packing parameter of the lipids. These results suggest that the shape of the compartment where a molecule is located plays a major role in controlling the chemical reactivity of non-enzymatic reactions. Moreover, the mathematical treatment we propose offers a useful tool for the design of vesicles with predictable reaction rates of the confined molecules, e.g., drug delivery vesicles with confined prodrugs. The results also show that a crude form of signal transduction, devoid of complex biological machinery, can be achieved by any external stimuli that drastically changes the structure of the membrane, like the osmotic shocks used in the present work.

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In living cells, communication requires the action of membrane receptors that are activated following very small environmental changes. A binary all-or-nothing behavior follows, making the organism extremely efficient at responding to specific stimuli. Using a minimal system composed of lipid vesicles, chemical models of a membrane receptor and their ligands, we show that bio-mimetic ON/OFF assembly of high avidity, multivalent domains is triggered by small temperature changes. Moreover, the intensity of the ON signal at the onset of the switch is modulated by the presence of small, weakly binding divalent ligands, reminiscent of the action of primary messengers in biological systems. Based on the analysis of spectroscopic data, we develop a mathematical model that rigorously describes the temperature-dependent switching of the membrane receptor assembly and ligand binding. From this we derive an equation that predicts the intensity of the modulation of the ON signal by the ligand-messenger as a function of the pairwise binding parameters, the number of binding sites that it features and the concentration. The behavior of our system, and the model derived, highlight the usefulness of weakly binding ligands in the regulation of membrane receptors and the pitfalls inherent to their binding promiscuity, such as non-specific binding to the membrane. Our model, and the equations derived from it, offer a valuable tool for the study of membrane receptors in both biological and biomimetic settings. The latter can be exploited to program membrane receptor avidity on sensing vesicles, create hierarchical protocell tissues or develop highly specific drug delivery vehicles.

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Using a combination of NMR and fluorescence measurements, we have investigated the structure and dynamics of the complexes formed between calcium-loaded calmodulin (CaM) and the potent breast cancer inhibitor idoxifene, a derivative of tamoxifen. High-affinity binding (Kd∼300 nM) saturates with a 2:1 idoxifene:CaM complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower-affinity antagonists TFP, J-8, and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular nuclear Overhauser effect (NOE) restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity, allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the 2:1 idoxifene:CaM complex illustrates how high-affinity binding occurs without the loss of extensive positional dynamics.

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Understanding self-assembly in confined spaces is essential to fully understand molecular processes in confined cell compartments and will offer clues on the behaviour of simple confined systems, such as protocells and lipid-vesicle based devices. Using a model system composed of lipid vesicles, a membrane impermeable receptor and a membrane-permeable ligand, we have studied in detail how compartmentalization modulates the interaction between the confined receptor and its ligand. We demonstrate that confinement of one of the building blocks stabilizes complex self-assembled structures to the extent that dilution leads, counterintuitively, to the formation of long range assemblies. The behaviour of the system can be explained by considering a confinement factor that is analogous, although not identical, to the effective molarity for intramolecular binding events. The confinement effect renders complex self-assembled species robust and persistent under conditions where they do not form in bulk solution. Moreover, we show that the formation of stable complex assemblies in systems compartmentalized by semi-permeable membranes does not require the prior confinement of all components, but only that of key membrane impermeable building blocks. To use a macroscopic analogy, lipid vesicles are like ship-in-a bottle constructs that are capable of directing the assembly of the confined ship following the confinement of a few key wooden planks. Therefore, we believe that the confinement effect described here would have played an important role in shaping the increase of chemical complexity within protocells during the first stages of abiogenesis. Additionally, we argue that this effect can be exploited to design increasingly efficient functional devices based on comparatively simple vesicles for applications in biosensing, nanoreactors and drug delivery vehicles.

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It is highly desirable that supramolecular polymers self-assemble following small changes in the environment. The degree of responsiveness depends on the degree of cooperativity at play during the assembly. Understanding how to modulate and quantify cooperativity is therefore highly desirable for the study and design of responsive polymers. Here we show that the cooperative assembly of a porphyrin-based, double-stranded polymer is triggered by changes in building blocks and in salt concentration. We develop a model that accounts for this responsiveness by assuming the binding of the salt countercations to the double-stranded polymer. Using our assembly model we generate plots that show the increase in concentration of polymer versus the normalized concentration of monomer. These plots are ideally suited to appreciate changes in cooperativity, and show that, for our system, these changes are consistent with the increase in polymer length observed experimentally. Unexpectedly, we find that polymer stability increases when cooperativity decreases. We attribute this behaviour to the fact that increasing salt concentration stabilizes the overall polymer more than the nucleus. In other words, the cooperativity factor α, defined as the ratio between the growth constant Kg and the nucleation constant Kn decreases as the overall stability of the polymer increases. Using our model to simulate the data, we generate cooperativity plots to explore changes in cooperativity for multistranded polymers. We find that, for the same pairwise association constants, the cooperativity sharply increases with the number of strands in the polymer. We attribute this dependence to the fact that the larger the number of strands, the larger is the nucleus necessary to trigger polymer growth. We show therefore that the cooperativity factor α does not properly account for the cooperativity behaviour of multistranded polymers, or any supramolecular polymer with a nucleus composed of more than 2 building blocks, and propose the use of the corrected cooperativity factor αm. Finally, we show that multistranded polymers display highly cooperative polymerisation with pairwise association constants as low as 10 M-1 between the building blocks, which should simplify the design of responsive supramolecular polymers.

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All-or-nothing molecular assembly events, essential for the efficient regulation of living systems at the molecular level, are emerging properties of complex chemical systems that are largely attributed to the cooperativity of weak interactions. The link between the self-assembly and the interactions responsible for the assembly is however often poorly defined. In this work we demonstrate how the chelate effect (multivalence cooperativity) can play a central role in the regulation of the all-or-nothing assembly of structures (supramolecular polymers here), even if the building blocks are not multivalent. We have studied the formation of double-stranded supramolecular polymers formed from Co-metalloporphyrin and bi-pyridine building blocks. Their cooperative nucleation-elongation assembly can be summarized as a thermodynamic cycle, where the monomer weakly oligomerizes linearly or weakly dimerizes laterally. But thanks to the chelate effect, the lateral dimer readily oligomerizes linearly and the oligomer readily dimerizes laterally, leading to long double stranded polymers. A model based on this simple thermodynamic cycle can be applied to the assembly of polymers with any number of strands, and allows for the determination of the length of the polymer and the all-or-nothing switching concentration from the pairwise binding constants. The model, which is consistent with the behaviour of supramolecular polymers such as microtubules and gelators, clearly shows that all-or-nothing assembly is triggered by a change in the mode of assembly, from non-multivalent to multivalent, when a critical concentration is reached. We believe this model is applicable to many molecular assembly processes, ranging from the formation of cell-cell focal adhesion points to crystallization.

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Although protein folding is often described by motion on a funnel-shaped overall topology of the energy landscape, the many local interactions that can occur result in considerable landscape roughness which slows folding by increasing internal friction. Recent experimental results have brought to light that this roughness also causes unusual diffusional behaviour of the backbone of an unfolded protein, i.e. the relative motion of protein sections cannot be described by the normal diffusion equation, but shows strongly subdiffusional behaviour with a nonlinear time dependence of the mean square displacement, 〈r(2)(t)〉∝t(α) (α≪ 1). This results in significantly slower configurational equilibration than had been assumed hitherto. Analysis of the results also allows quantification of the energy landscape roughness, i.e. the root-mean-squared depth of local minima, yielding a value of 4-5kBT for a typical small protein.

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A self-assembled nanoparticle containing a photosensitizer and a Trojan-horse moiety (cholesterol), binds an anti-TB pro-drug and increases 1000-fold its activity against mycobacteria. These minimalist constructs will allow development of economically viable, efficient drug preparations for the treatment of drug-resistant TB infections.

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In living cells and biomimetic systems alike, multivalent ligands in solution can induce clustering of membrane receptors. The link between the receptor clustering and the ligand binding remains, however, poorly defined. Using minimalist divalent ligands, we develop a model that allows quantifying the modulation of receptor clustering by binding of ligands with any number of binding sites. The ligands, with weak binding affinity for the receptor and with binding sites held together by flexible linkers, lead to nearly quantitative clustering upon binding in a wide range of experimental conditions, showing that efficient modulation of receptor clustering does not require pre-organization or large binding affinities per binding site. Simulations show that, in the presence of ligands with five or more binding sites, an on/off clustering response follows a very small change in receptor density in the membrane, which is consistent with the highly cooperative behavior of multivalent biomolecular systems.

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The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4-5 k(B)T). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 k(B)T) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues.

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Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer's, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from ß(2)-microglobulin (ß(2)m). Using cryoelectron tomography, we demonstrate that fragmented ß(2)m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15-25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid-protein interactions in membrane remodelling.

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Multiresponsive low-molecular-weight hydrogelators (LMWHs) are ideal candidates for the development of smart, soft, nanotechnology materials. The synthesis is however very challenging. On the one hand, de novo design is hampered by our limited ability to predict the assembly of small molecules in water. On the other hand, modification of pre-existing LMWHs is limited by the number of different stimuli-sensitive chemical moieties that can be introduced into a small molecule without seriously disrupting the ability to gelate water. Herein we report the synthesis and characterization of multistimuli LMWHs, based on a modular design, composed of a hydrophobic, disulfide, aromatic moiety, a maleimide linker, and a hydrophilic section based on an amino acid, here N-acetyl-L-cysteine (NAC). As most LMWHs, these gelators experience reversible gel-to-sol transition following temperature changes. Additionally, the NAC moiety allows reversible control of the assembly of the gel by pH changes. The reduction of the aromatic disulfide triggers a gel-to-sol transition that, depending on the design of the particular LMWH, can be reverted by reoxidation of the resulting thiol. Finally, the hydrolysis of the cyclic imide moieties provides an additional trigger for the gel-to-sol transition with a timescale that is appropriate for use in drug-delivery applications. The efficient response to the multiple external stimuli, coupled to the modular design makes these LMWHs an excellent starting point for the development of smart nanomaterials with applications that include controlled drug release. These hydrogelators, which were discovered by serendipity rather than design, suggest nonetheless a general strategy for the introduction of multiple stimuli-sensitive chemical moieties, to offset the introduction of hydrophilic moieties with additional hydrophobic ones, in order to minimize the upsetting of the critical hydrophobic-hydrophilic balance of the LMWH.

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Thanks largely to a cooperative chelate effect, clustered membrane-embedded proteins favourably bind to multivalent ligands in solution and, conversely, a multivalent receptor can induce the clustering of membrane-embedded proteins. Here, we use a chemical model to show that the binding of a monovalent ligand and the clustering of a membrane-embedded receptor are closely related processes that modulate each other without the contribution of any apparent multivalence effect. Clearly, the confinement of the receptor within the surface reveals cooperative effects between clustering and binding that are too weak to detect in bulk-solution systems. This work shows that for membrane-embedded receptors that undergo some degree of spontaneous clustering, analyses based on multivalence-mediated cooperativity are insufficient to describe fully the molecular recognition events induced by ligands in solution. Instead, a binding-clustering thermodynamic cycle is proposed for the analysis of the interaction of any kind of ligand with membrane-embedded receptors.

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Biomolecular and artificial receptors are typically designed to exploit the hydrophobic effect in order to enhance the stability of receptor-ligand complexes in water. For example, artificial receptors are often built around hydrophobic cavities. These receptors exploit the hydrophobic effect toward ligand recognition, but the structure of the binding site requires a rigid framework to overcome the hydrophobic effect-driven tendency to collapse. Here we present an artificial receptor that exploits the hydrophobic effect to define its structure in water. The receptor is based on amphiphilic building blocks that assemble into micelle-like aggregates of a very high stability, attributed to the unusual shape of the amphiphile: a relatively rigid molecule composed of a large hydrophobic segment, based on the cholesterol molecule, and a very large headgroup build around a Zn-metalloporphyrin moiety. The assemblies, persistent down to the nanomolar range, are better described as self-assembled nanoparticles. Within the nanoparticle-water interface, Zn-metalloporphyrin moieties form multiple binding sites that specifically bind ligands bearing basic nitrogen atoms. The nanoparticles show enhanced binding affinity relative to a model receptor that does not self-assemble. Structurally related ligands show a correlation between the enhancement of binding and the octanol/water partition coefficient, log P, suggesting that the desolvation of binding sites is the main driving force for the enhancement of binding affinity at the nanoparticle-water interface. In addition, the highest affinity observed for the ditopic ligands relative to the monotopic ligands is evidence of a multivalent effect operating within this type of receptors. The nanoparticle readily deassembles upon addition of water-miscible organic solvents, such as methanol, or in the presence of detergents. This approach toward self-assembled receptors can be easily adapted to the development of differential receptors by the simple expedient of mixing slightly different amphiphiles (i.e., different metals in the porphyrin ring for the amphiphiles described here) in variable proportions.

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High-dilution equilibrium macrocyclization is developed as a general approach to trapping proteins in a non-native state with a synthetic cross-linking agent. The approach is illustrated using the N-terminal domain of phosphoglycerate kinase and a synthetic reagent containing two maleimide groups, for selective attachment to cysteines introduced onto the protein surface through mutagenesis, and an aromatic disulfide that can be chemically or photochemically cleaved. Following functionalization of the cysteine residues, thiol-disulfide exchange chemistry under strongly unfolding conditions was used to achieve intramolecular cyclization and a high yield of the cross-linked protein. (1)H NMR, CD, and fluorescence spectroscopies indicate that the conformation of the cross-linked protein is non-native. Chemical cleavage of the aromatic disulfide cross-link by a reducing agent results in the acquisition of a nativelike conformation for the reduced protein. Thus, the cross-link acts as a reversible switch of protein folding.

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The essential thiol of the enzyme papain has been caged by linking to an aromatic thiol. The resulting caged protein is inactive but enzymatic activity is fully restored upon chemical cleavage of the protective disulfide bond. We have exploited the chemistry of this disulfide bond to uncage papain by pulse radiolysis. We have shown that up to 10% of the enzyme activity can be restored by reductive pulse radiolysis. This approach has been tested on a small-molecule model system, and experiments on this model compound show that pulse radiolysis of the mixed cysteine-aromatic disulfide results in selective reduction of the disulfide bond to generate a thiol in 10-20% yield, consistent with the radiolytically restored activity of the caged papain quantified by the biochemical assay.

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A well known strategy to prepare high affinity ligands for a biological receptor is to link together low affinity ligands. DCC (dynamic combinatorial chemistry) was used to select bifunctional protein ligands with high affinity relative to the corresponding monofunctional ligands. Thiol to disulfide linkage generated a small dynamic library of bifunctional ligands in the presence of calmodulin, a protein with two independently mobile domains. The binding constant of the bifunctional ligand (disulfide) most amplified by the presence of calmodulin is nearly two orders of magnitude higher than that of the corresponding monofunctional ligand (thiol).

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Using calmodulin antagonism as a model, it is demonstrated that, under circumstances in which binding sites are motionally independent, it is possible to create bifunctional ligands that bind with significant affinity enhancement over their monofunctional counterparts. Suitable head groups were identified by using a semiquantitative screen of monofunctional tryptophan analogs. Two bifunctional ligands, which contained two copies of the highest-affinity head group tethered by rigid linkers, were synthesized. The bifunctional ligands bound to calmodulin with a stoichiometry of 1:1 and with an affinity enhancement over their monofunctional counterparts; the latter bound with a stoichiometry of 2:1 ligand:protein. A lower limit to the effective concentrations of the domains of calmodulin relative to each other (0.2-2 mM) was determined. A comparable effective concentration was achieved for bifunctional ligands based on higher-affinity naphthalene sulphonamide derivatives.

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A new series of photocleavable protein cross-linking reagents based on bis(maleimide) derivatives of diaryl disulfides have been synthesised. They have been functionalised with cysteine and transient absorption spectra for the photolysis reaction have been recorded by using the pump-probe technique with a time resolution of 100 femtoseconds. Photolysis of the disulfide bond yields the corresponding thiyl radicals in less than a picosecond. There is a significant amount of geminate recombination, but some of the radicals escape the solvent cage and the quantum yield for photocleavage is 30 % in water.

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