Solvent-Dependent Structural Dynamics in the Ultrafast Photodissociation Reaction of Triiodide Observed with Time-Resolved X-ray Solution Scattering.

Resolving the structural dynamics of bond breaking, bond formation, and solvation is required for a deeper understanding of solution-phase chemical reactions. In this work, we investigate the photodissociation of triiodide in four solvents using femtosecond time-resolved X-ray solution scattering following 400 nm photoexcitation. Structural analysis of the scattering data resolves the solvent-dependent structural evolution during the bond cleavage, internal rearrangements, solvent-cage escape, and bond reformation in real time. The nature and structure of the reaction intermediates during the recombination are determined, elucidating the full mechanism of photodissociation and recombination on ultrafast time scales. We resolve the structure of the precursor state for recombination as a geminate pair. Further, we determine the size of the solvent cages from the refined structures of the radical pair. The observed structural dynamics present a comprehensive picture of the solvent influence on structure and dynamics of dissociation reactions.

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

Membrane permeabilization can be crucially biased by a fusogenic lipid composition - leaky fusion caused by antimicrobial peptides in model membranes.

Induced membrane permeabilization or leakage is often taken as an indication for activity of membrane-active molecules, such as antimicrobial peptides (AMPs). The exact leakage mechanism is often unknown, but important, because certain mechanisms might actually contribute to microbial killing, while others are unselective, or potentially irrelevant in an in vivo situation. Using an antimicrobial example peptide (cR3W3), we illustrate one of the potentially misleading leakage mechanisms: leaky fusion, where leakage is coupled to membrane fusion. Like many others, we examine peptide-induced leakage in model vesicles consisting of binary mixtures of anionic and zwitterionic phospholipids. In fact, phosphatidylglycerol and phosphatidylethanolamine (PG/PE) are supposed to reflect bacterial membranes, but exhibit a high propensity for vesicle aggregation and fusion. We describe the implications of this vesicle fusion and aggregation for the reliability of model studies. The ambiguous role of the relatively fusogenic PE-lipids becomes clear as leakage decreases significantly when aggregation and fusion are prevented by sterical shielding. Furthermore, the mechanism of leakage changes if PE is exchanged for phosphatidylcholine (PC). We thus point out that the lipid composition of model membranes can be biased towards leaky fusion. This can lead to discrepancies between model studies and activity in true microbes, because leaky fusion is likely prevented by bacterial peptidoglycan layers. In conclusion, choosing the model membrane might implicate the type of effect (here leakage mechanism) that is observed. In the worst case, as with leaky fusion of PG/PE vesicles, this is not directly relevant for the intended antimicrobial application.

Interplay of Fusion, Leakage, and Electrostatic Lipid Clustering: Membrane Perturbations by a Hydrophobic Antimicrobial Polycation.

Membrane active compounds are able to induce various types of membrane perturbations. Natural or biomimetic candidates for antimicrobial treatment or drug delivery scenarios are mostly designed and tested for their ability to induce membrane permeabilization, also termed leakage. Furthermore, the interaction of these usually cationic amphiphiles with negatively charged vesicles often causes colloidal instability leading to vesicle aggregation or/and vesicle fusion. We show the interplay of these modes of membrane perturbation in mixed phosphatidyl glycerol (PG)/phosphatidyl ethanolamine (PE) by the statistical copolymer MM:CO comprising, both, charged and hydrophobic subunits. MM:CO is a representative of partially hydrophobic, highly active, but less selective antimicrobial polycations. Cryo-electron microscopy indicates vesicle fusion rather than vesicle aggregation upon the addition of MM:CO to negatively charged PG/PE (1:1) vesicles. In a combination of fluorescence-based leakage and fusion assays, there is support for membrane permeabilization and pronounced vesicle fusion activity as distinct effects. To this end, membrane fusion and aggregation were prevented by including lipids with polyethylene glycol attached to their head groups (PEG-lipids). The leakage activity of MM:CO is very similar in the absence and presence of PEG-lipids. Vesicle aggregation and fusion however are largely suppressed. This strongly suggests that MM:CO induces leakage by asymmetric packing stress because of hydrophobically driven interactions which could lead to leakage. As a further membrane perturbation effect, MM:CO causes lipid clustering in model vesicles. We address potential artifacts and misinterpretations of experiments characterizing leakage and fusion. Additional to the leakage activity, the pronounced fusogenic activity of the polymer and potentially of many other similar compounds likely has implications for antimicrobial activity and beyond.

Hidden complexity in membrane permeabilization behavior of antimicrobial polycations.

A promising alternative to classical antibiotics are antimicrobial peptides and their synthetic mimics (smAMPs) that supposedly act directly on membranes. For a more successful design of smAMPs, we need to know how the type of interaction with the membrane determines the type of membrane perturbation. How this, in turn, transfers into selectivity and microbial killing activity is largely unknown. Here, we characterize the action of two smAMPs: MM:CO (a copolymer of hydrophobic cyclooctyl subunits and charged ß-monomethyl-α-aminomethyl subunits) and the highly charged poly-NM (a homopolymer of α-aminomethyl subunits). By thorough characterization of vesicle leakage experiments, we elucidate complex membrane perturbation behavior in zwitterionic or negatively charged vesicles. Vesicle leakage data does not entirely agree with the growth inhibition of microbes. Our ensemble of advanced membrane permeabilization approaches clarifies these discrepancies. Long cumulative leakage kinetics show that the two smAMPs act either by transient leakage or by rare stochastic leakage events that occur at charge neutralization in the sample. We determine the strengths of individual leakage events induced by the smAMPs in membranes of various compositions. These strengths indicate changes in leakage mechanism over time and concentration range. Thus, our sophisticated analysis of vesicle leakage experiments reveals a fine-tuned flexibility in membrane permeabilization mechanisms. These details are indispensable in judging and designing membrane-active compounds.

Signal amplification and transduction in phytochrome photosensors.

Sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains. Phytochromes are a major family of red-light-sensing kinases that control diverse cellular functions in plants, bacteria and fungi. Bacterial phytochromes consist of a photosensory core and a carboxy-terminal regulatory domain. Structures of photosensory cores are reported in the resting state and conformational responses to light activation have been proposed in the vicinity of the chromophore. However, the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive. Here we report crystal and solution structures of the resting and activated states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans. The structures show an open and closed form of the dimeric protein for the activated and resting states, respectively. This nanometre-scale rearrangement is controlled by refolding of an evolutionarily conserved 'tongue', which is in contact with the chromophore. The findings reveal an unusual mechanism in which atomic-scale conformational changes around the chromophore are first amplified into an ångstrom-scale distance change in the tongue, and further grow into a nanometre-scale conformational signal. The structural mechanism is a blueprint for understanding how phytochromes connect to the cellular signalling network.

EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation.

The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.

Quantified Membrane Permeabilization Indicates the Lipid Selectivity of Membrane-Active Antimicrobials.

Most antimicrobial peptides (AMPs) and their synthetic mimics (SMAMPs) are thought to act by permeabilizing cell membranes. For antimicrobial therapy, selectivity for pathogens over mammalian cells is a key requirement. Understanding membrane selectivity is thus essential for designing AMPs and SMAMPs to complement classical antibiotics in the future. This study focuses on membrane permeabilization induced by SMAMPs and their selectivity for membranes with different lipid compositions. We measure release and fluorescence lifetime of a self-quenching dye in lipid vesicles. Apart from the dose-response, we quantify the strength of individual leakage events, and, employing cumulative kinetics, categorize permeabilization behavior. We propose that differing selectivities in a series of SMAMPs arise from a combination of the effect of the antimicrobial agent and the susceptibility of the membrane (with a given lipid composition) for certain types of leakage behavior. The unselective and hemolytic SMAMP is found to act mainly by the asymmetry stress mechanism, mediated by hydrophobic insertion of SMAMPs into lipid layers. The more selective SMAMPs induced leakage events occurring stochastically over several hours. Lipid intrinsic properties might additionally amplify the efficiency of leakage events. Leakage behavior changes with both the design of the SMAMP and the lipid composition of the membrane. Understanding how leakage behavior contributes to the selectivity and activity of antimicrobial agents will aid the design and screening of antimicrobials. An understanding of the underlying processes facilitates the comparison of membrane permeabilization across in vitro and in vivo assays.

Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: Complex interplay of electrostatic and hydrophobic interactions.

Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely omithine (Orn), alpha, gamma-diaminobutyric acid (Dab) and alpha, beta-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer.

Leaky membrane fusion: an ambivalent effect induced by antimicrobial polycations.

Both antimicrobial peptides and their synthetic mimics are potential alternatives to classical antibiotics. They can induce several membrane perturbations including permeabilization. Especially in model studies, aggregation of vesicles by such polycations is often reported. Here, we show that unintended vesicle aggregation or indeed fusion can cause apparent leakage in model studies that is not possible in most microbes, thus potentially leading to misinterpretations. The interactions of a highly charged and highly selective membrane-active polycation with negatively charged phosphatidylethanolamine/phosphatidylglycerol (PE/PG) vesicles are studied by a combination of biophysical methods. At low polycation concentrations, apparent vesicle aggregation was found to involve exchange of lipids. Upon neutralization of the negatively charged vesicles by the polycation, full fusion and leakage occurred and leaky fusion is suspected. To elucidate the interplay of leakage and fusion, we prevented membrane contacts by decorating the vesicles with PEG-chains. This inhibited fusion and also leakage activity. Leaky fusion is further corroborated by increased leakage with increasing likeliness of vesicle-vesicle contacts. Because of its similar appearance to other leakage mechanisms, leaky fusion is difficult to identify and might be overlooked and more common amongst polycationic membrane-active compounds. Regarding biological activity, leaky fusion needs to be carefully distinguished from other membrane permeabilization mechanisms, as it may be less relevant to bacteria, but potentially relevant for fungi. Furthermore, leaky fusion is an interesting effect that could help in endosomal escape for drug delivery. A comprehensive step-by-step protocol for membrane permeabilization/vesicle leakage using calcein fluorescence lifetime is provided in the ESI.

Tracking the Endosomal Escape: A Closer Look at Calcein and Related Reporters.

Crossing the cellular membrane and delivering active pharmaceuticals or biologicals into the cytosol of cells is an essential step in the development of nanomedicines. One of the most important intracellular processes regarding the cellular uptake of biologicals is the endolysosomal pathway. Sophisticated nanocarriers are developed to overcome a major hurdle, the endosomal entrapment, and delivering their cargo to the required site of action. In parallel, in vitro assays are established analyzing the performance of these nanocarriers. Among them, the release of the membrane-impermeable dye calcein has become a popular and straightforward method. It is accessible for most researchers worldwide, allows for rapid conclusions about the release potential, and enables the study of release mechanisms. This review is intended to provide an overview and guidance for scientists applying the calcein release assay. It comprises a survey of several applications in the study of endosomal escape, considerations of potential pitfalls, challenges, and limitations of the assay, and a brief summary of complementary methods. Based on this review, it is hoped to encourage further research groups to take advantage of the calcein release assay for their own purposes and help to create a database for more efficient cross-correlations between nanocarriers.

Triggers for ß-sheet formation at the hydrophobic-hydrophilic interface: high concentration, in-plane orientational order, and metal ion complexation.

Amyloid formation plays a causative role in neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. Soluble peptides form ß-sheets that subsequently rearrange into fibrils and deposit as amyloid plaques. Many parameters trigger and influence the onset of the ß-sheet formation. Early stages are recently discussed to be cell-toxic. Aiming at understanding various triggers such as interactions with hydrophobic-hydrophilic interfaces and metal ion complexation and their interplay, we investigated a set of model peptides at the air-water interface. We are using a general approach to a variety of diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes that are connected to amyloid formation. Surface sensitive techniques combined with film balance measurements have been used to assess the conformation of the peptides and their orientation at the air-water interface (IR reflection-absorption spectroscopy). Additionally, the structures of the peptide layers were characterized by grazing incidence X-ray diffraction and X-ray reflectivity. The peptides adsorb to the air-water interface and immediately adopt an α-helical conformation. This helical intermediate transforms into ß-sheets upon further triggering. The factors that result in ß-sheet formation are dependent on the peptide sequence. In general, the interface has the strongest effect on peptide conformation compared to high concentrations or metal ions. Metal ions are able to prevent aggregation in bulk but not at the interface. At the interface, metal ion complexation has only minor effects on the peptide secondary structure, influencing the in-plane structure that is formed in two dimensions. At the air-water interface, increased concentrations or a parallel arrangement of the α-helical intermediates are the most effective triggers. This study reveals the role of various triggers for ß-sheet formation and their complex interplay. Our main finding is that the hydrophobic-hydrophilic interface largely governs the conformation of peptides. Therefore, the present study implies that special care is needed when interpreting data that may be affected by different amounts or types of interfaces during experimentation.

Biomembrane Permeabilization: Statistics of Individual Leakage Events Harmonize the Interpretation of Vesicle Leakage.

The mode of action of membrane-active molecules, such as antimicrobial, anticancer, cell penetrating, and fusion peptides and their synthetic mimics, transfection agents, drug permeation enhancers, and biological signaling molecules (e.g., quorum sensing), involves either the general or local destabilization of the target membrane or the formation of defined, rather stable pores. Some effects aim at killing the cell, while others need to be limited in space and time to avoid serious damage. Biological tests reveal translocation of compounds and cell death but do not provide a detailed, mechanistic, and quantitative understanding of the modes of action and their molecular basis. Model membrane studies of membrane leakage have been used for decades to tackle this issue, but their interpretation in terms of biology has remained challenging and often quite limited. Here we compare two recent, powerful protocols to study model membrane leakage: the microscopic detection of dye influx into giant liposomes and time-correlated single photon counting experiments to characterize dye efflux from large unilamellar vesicles. A statistical treatment of both data sets does not only harmonize apparent discrepancies but also makes us aware of principal issues that have been confusing the interpretation of model membrane leakage data so far. Moreover, our study reveals a fundamental difference between nano- and microscale systems that needs to be taken into account when conclusions about microscale objects, such as cells, are drawn from nanoscale models.

Sequential conformational transitions and α-helical supercoiling regulate a sensor histidine kinase.

Sensor histidine kinases are central to sensing in bacteria and in plants. They usually contain sensor, linker, and kinase modules and the structure of many of these components is known. However, it is unclear how the kinase module is structurally regulated. Here, we use nano- to millisecond time-resolved X-ray scattering to visualize the solution structural changes that occur when the light-sensitive model histidine kinase YF1 is activated by blue light. We find that the coiled coil linker and the attached histidine kinase domains undergo a left handed rotation within microseconds. In a much slower second step, the kinase domains rearrange internally. This structural mechanism presents a template for signal transduction in sensor histidine kinases.Sensor histidine kinases (SHK) consist of sensor, linker and kinase modules and different models for SHK signal transduction have been proposed. Here the authors present nano- to millisecond time-resolved X-ray scattering measurements, which reveal a structural mechanism for kinase domain activation in SHK.

Membrane binding of peptide models for early stages of amyloid formation: Lipid packing counts more than charge.

Amyloid formation is related to neurodegenerative diseases like Alzheimer's disease or Parkinson's disease. In the molecular onset of the diseases, soluble peptides adopt conformations that are rich in ß-sheet and ultimately form aggregates. How this process is triggered or influenced by membrane binding, or how the membrane integrity is disturbed by the peptide binding and conformational transition is still under debate. In the present study, we systematically examine the effects of ß-sheet prone model peptides on zwitterionic and negatively charged lipids in both mono- and bilayers and in various lipid phase states by infrared reflection absorption spectroscopy, grazing incidence X-ray diffraction, and small and wide angle X-ray scattering. No difference in the interaction of the peptides with zwitterionic or negatively charged lipids was observed. Furthermore, the interaction of ß-sheet prone model peptides leaves the lipid structure largely unaffected. However, the lipid phase state decides upon the mode of interaction. Peptides insert into liquid-expanded layers and interact only with the head groups of liquid-condensed lipid layers. Using a zoo of complementary techniques and critically examining preparation procedures we are able to obtain an unambiguous picture of peptide binding to membranes.

Light-induced structural changes in a monomeric bacteriophytochrome.

Phytochromes sense red light in plants and various microorganism. Light absorption causes structural changes within the protein, which alter its biochemical activity. Bacterial phytochromes are dimeric proteins, but the functional relevance of this arrangement remains unclear. Here, we use time-resolved X-ray scattering to reveal the solution structural change of a monomeric variant of the photosensory core module of the phytochrome from Deinococcus radiodurans. The data reveal two motions, a bend and a twist of the PHY domain with respect to the chromophore-binding domains. Infrared spectroscopy shows the refolding of the PHY tongue. We conclude that a monomer of the phytochrome photosensory core is sufficient to perform the light-induced structural changes. This implies that allosteric cooperation with the other monomer is not needed for structural activation. The dimeric arrangement may instead be intrinsic to the biochemical output domains of bacterial phytochromes.

Structural photoactivation of a full-length bacterial phytochrome.

Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes.

Ubiquitous Structural Signaling in Bacterial Phytochromes.

The phytochrome family of light-switchable proteins has long been studied by biochemical, spectroscopic and crystallographic means, while a direct probe for global conformational signal propagation has been lacking. Using solution X-ray scattering, we find that the photosensory cores of several bacterial phytochromes undergo similar large-scale structural changes upon red-light excitation. The data establish that phytochromes with ordinary and inverted photocycles share a structural signaling mechanism and that a particular conserved histidine, previously proposed to be involved in signal propagation, in fact tunes photoresponse.