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The following theses are claimed, several contrasting current climate policies and taxonomies. Analysis, based on solely carbon dioxide emission and energy budget, concludes a set of concrete solutions for mitigating climate change effects. Some of the theses violate more orthodox policy which is thus protested against in order to move forward. • Our long-term goal must be to stop using all carbon-containing fuels, including natural gas and other fossil products as well as biofuels. • We must electrify society and industry, with electricity from only non-carbon-based power including nuclear power, hydro-electric, wind and solar power. • We must prepare ourselves for changes. Even if the present emission volumes of carbon dioxide were possible to stop immediately, various lag effects are inevitable and negative development will therefore continue for considerable time. • We must count with continued melting of land ice, the complete liquifying of the Antarctica ice expected to lead to a global sea level rise by some 60 m, flooding most capitals. Among various solutions to mitigate the effects of ice melting, including lowered global temperatures, the following is proposed. • To mitigate sea level rise, stationary water reservoirs should be built around the world. With estimated melting rates it would require ca 1 million reservoirs be deployed or expanded during the next 20-40 years. • Such reservoirs could also solve the emergent problem of lack of fresh water in many places. They could also be used for local storage of hydroelectric energy by using pump storage hydroelectric (PSH) technology. • All energy production sources should be analyzed according to a Total Balanced Energy Budget (TBEB) with the main objective of minimizing the emissions of greenhouse gases. • For each region/country, a table of available or conceivable complementary electric energy sources should be made and ranked according to TBEB—the sources given priority weights depending on feasibility, significance, and environmental friendliness. Tables are presented for Sweden, Norway, Denmark, Germany, France, Ukraine, California, Massachusetts, Maine, Peru, Australia, China and Japan. Generally, we find the following rank of priority applicable. • Solar energy from desert arid areas is given highest priority in replacing carbon-based forms of energy. Submarine electric cables may be deployed along the Australia-Singapore model, if the available power grids are insufficient for the energy transport. • Electrolysis of water producing clean hydrogen gas is given very high priorityboth for using hydrogen as fuel as well as for energy storage. Improved efficiency should be achieved by the development of electrolysis catalysts. • Hydroelectric power in combination with PSH is given high priority to mitigate both grid power fluctuations as well as source (solar and wind) intermittence. • False hope should not be seeded among society and politicians by inflating projects that are less realistic or suboptimal for technological, economic or other reasons. Here, probably most forms of “biofuels” (which although being “carbon neutral” do produce carbon dioxide) and “carbon capture” (catching carbon dioxide gas at the combustion site, compressing it to liquid and depositing it in salt mines or empty oil fields) are considered less significant compared to other more direct solutions. Both biofuels and carbon capture may be associated with social and environmental issues. • Political legislation and instruments (“taxonomy”) invented with the original objective of mitigating negative climate change effects should be reanalyzed and changed if not functional. The EU Emissions Trading System (EU ETS)—a market for outlet rights, for example, is a local initiative which despite its valuable ambition might be suboptimal with respect to goal of efficient decrease of carbon dioxide emission globally. Similarly, “climate taxonomy” can create loopholes bypassing a sound TBEB. • Science-based targets (SBT) to decarbonize the private sector as part of global efforts to achieve the temperature goal of the Paris Agreement should be further encouraged. • Solve economic and political challenges allowing and promoting establishment of required international energy collaborations (e.g., for solar energy cross-continental transport programs).

The neuronal protein α-synuclein, linked to Parkinson’s disease, binds to negatively charged vesicles adopting a partial α-helix structure, but helix arrangement at the vesicle surface is not fully understood. Using linear dichroism spectroscopy (LD), we study the interaction of monomeric α-synuclein with large unilamellar vesicles of 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), and 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) under mild shear flow. The LD data of oriented lipid vesicles show that the long axis of the protein helix is oriented preferentially perpendicular to the membrane normal but deviates from a uniform in-plane distribution. Upon initial binding, a fraction of helices are oriented in the direction of least curvature for all ellipsoid-shaped vesicles at a lipid:protein molar ratio of 100. However, at a lower protein concentration the helices distribute uniformly on DOPS and POPS vesicles. In all cases, the α-synuclein helices rearrange with time (minute time scale) in the shear flow and begin to tilt into the vesicle membrane. Faster reorientation kinetics in the presence of flow suggests that modulation of membrane dynamics, by thermal or shear-dynamic activation, may overcome steric barriers by what may be called “flow catalysis”.

The mole a concept and Avogadro’s number are discussed as sought by Albert Einstein in his PhD thesis of 1905. Einstein would probably have regarded the metric system of units based on centimetre-gram-second (cgs) preferable to today’s SI system and specifically he would have rejected a recent SI suggestion to redefine Avogadro’s constant as based on a nonatomistic continuum description of matter. He would probably also have preferred keeping a dualistic definition of mole able of bookkeeping both mass and number of particles: we advocate that here and call it the ‘Einstein Definition’ and as Avogadro’s number we shall adopt an integer, the cube of 84446888 as suggested by Fox and Hill, providing also a definition of the kilogram based on the atomic mass of the carbon 12 isotope. Einstein was the first to explain the microscopic movements of pollen grains reported by Robert Brown in 1828 and his explanation that the particles move as a result of an unequal number of water molecules bumping into them from opposite sides was what finally made the scientific world accept the atom theory in its modern shape. In a cosmic diffusion analogy, pollen or bacterial spores moving randomly in outer space driven by the solar winds between solar systems can be envisaged. Applying Einstein’s diffusion theory, one can argue that life might have emerged from far outside of our planet from billions of solar systems, though not from outside of our Milky Way galaxy. As a curiosity we note that the number of solar systems (stars) in the Universe has been estimated to be of the order of Avogadro’s number. a Etymology: “Mole” was derived from French Molécule (extremely small particle) or diminutive from Greek [Formula: see text] (molos= substance). In analogy with atom, derived from Greek atomos or atemnein, uncleavable, we may thus interpret molecule as the smallest part that a substance can be divided into without losing its chemical character.

In contrast to DNA replication and transcription where nucleotides are added and matched one by one, homologous recombination by DNA strand exchange tests whole sequences for complementarity, which requires elimination of mismatched yet thermodynamically stable intermediates. To understand the remarkable sequence specificity of homologous recombination, we have studied strand exchange between a 20-mer duplex containing one single mismatch (placed at varied positions) with the matching single strand in presence of poly(ethylene glycol) representing a semi-hydrophobic environment. A FRET-based assay shows that rates and yields of strand exchange from mismatched to matched strands rapidly increase with semi-hydrophobic co-solute concentration, contrasting previously observed general strand exchange accelerating effect of ethyl glycol ethers. We argue that this effect is not caused simply by DNA melting or solvent-induced changes of DNA conformation but is more complex involving several mechanisms. The catalytic effects, we propose, involve strand invasion facilitated by reduced duplex stability due to weakened base stacking ("longitudinal breathing"). Secondly, decreased water activity makes base-pair hydrogen bonds stronger, increasing the relative energy penalty per mismatch. Finally, unstacked mismatched bases (gaps) are stabilized through partly intercalated hydrophobic co-solvent molecules, assisting nucleation of strand invasion at the point of mismatch. We speculate that nature long ago discovered, and now exploits in various enzymes, that sequence recognition power of nucleic acids may be modulated in a hydrophobic environment.

The first two-photon entanglement experiment performed 50 years ago by Kocher and Commins (KC) provided isolated pairs of entangled photons from an atomic three-state fluorescence cascade. In view of questioning of Bell’s theorem, data from these experiments are re-analyzed and shown sufficiently precise to confirm quantum mechanical and dismiss semi-classical theory without need for Bell’s inequalities. Polarization photon correlation anisotropy (A) is useful: A is near unity as predicted quantum mechanically and well above the semi-classic range, 0 ⩽ A ⩽ 1 / 2 . Although yet to be found, one may envisage a three-state molecule emitting entangled photon pairs, in analogy with the KC atomic system. Antibunching in fluorescence from single molecules in matrix and entangled photons from quantum dots promise it be possible. Molecules can have advantages to parametric down-conversion as the latter photon distribution is Poissonian and unsuitable for producing isolated pairs of entangled photons. Analytical molecular applications of entangled light are also envisaged.

Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small β-sheets, the key motifs of proteins, undergoing structural changes in liquid–liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W → LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3–0.5 kcal·mol−1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2–2.3 kcal·mol−1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has &gt, 1 kcal·mol−1 per residue excess stabilization.

The cell membrane is an ordered environment, which anisotropically affects the structure and interactions of all of its molecules. Monitoring membrane orientation at a local level is rather challenging but could reward crucial information on protein conformation and interactions in the lipid bilayer. We monitored local lipid ordering changes upon varying the cholesterol concentration using polarized light spectroscopy and pyrene as a membrane probe. Pyrene, with a shape intermediate between a disc and a rod, can detect microscopic orientation variations at the level of its size. The global membrane orientation was determined using curcumin, a probe with nonoverlapping absorption relative to that of pyrene. While the macroscopic orientation of a liquid-phase bilayer decreases with increasing cholesterol concentration, the local orientation is improved. Pyrene is found to be sensitive to the local effects induced by cholesterol and temperature on the bilayer. Disentangling local and global orientation effects in membranes could provide new insights into functionally significant interactions of membrane proteins.

Insulin and lysozyme share the common features of being prone to aggregate and having biomedical importance. Encapsulating lysozyme and insulin in micellar nanoparticles probably would prevent aggregation and facilitate oral drug delivery. Despite the vivid structural knowledge of lysozyme and insulin, the environment-dependent oligomerization (dimer, trimer, and multimer) and associated structural dynamics remain elusive. The knowledge of the intra- and intermolecular interaction profiles has cardinal importance for the design of encapsulation protocols. We have employed various biophysical methods such as NMR spectroscopy, X-ray crystallography, Thioflavin T fluorescence, and atomic force microscopy in conjugation with molecular modeling to improve the understanding of interaction dynamics during homo-oligomerization of lysozyme (human and hen egg) and insulin (porcine, human, and glargine). The results obtained depict the atomistic intra- and intermolecular interaction details of the homo-oligomerization and confirm the propensity to form fibrils. Taken together, the data accumulated and knowledge gained will further facilitate nanoparticle design and production with insulin or lysozyme-related protein encapsulation.

The assembly of stacked dyes on DNA is of interest for electron transfer, light harvesting, sensing, and catalysis applications. A combination of UV/vis absorption, linear dichroism (LD), and circular dichroism (CD) was applied to characterize thoroughly the aggregation with DNA of the phenothiazine dyes methylene blue, azure B, and thionine. Aggregates of each dye with [poly(dG-dC)]2, [poly(dA-dT)]2, and calf thymus DNA were explored at high dye:DNA binding ratios, where excess dye groove-binds after all intercalation sites are filled. The organization of the aggregates (dimers, trimers, and multimers) with polydeoxynucleotides displays a structural diversity that depends on DNA sequence, extent of methylation of dye exocyclic amine groups, and ionic strength. The dyes typically form right-handed H-aggregates having negative LD, consistent with stepped stacking along the minor groove. However, aggregates in some dye:DNA aggregates show left-handed chirality or positive LD, indicating unusual modes of agg...

To address the mechanistic roles of ATP hydrolysis in RecA-promoted strand exchange reaction in homologous recombination, quantum mechanical calculations are performed on key parts of the RecA-DNA complex. We find that ATP hydrolysis may induce changes at the protein-DNA interface, resulting in the rearrangement of the hydrogen bond network connecting the ATP and the DNA binding sites.

Interactions between human lysozyme (HL) and the lipopolysaccharide (LPS) of Klebsiella pneumoniae O1, a causative agent of lung infection, were identified by surface plasmon resonance. To characterize the molecular mechanism of this interaction, HL binding to synthetic disaccharides and tetrasaccharides representing one and two repeating units, respectively, of the O-chain of this LPS were studied. pH-dependent structural rearrangements of HL after interaction with the disaccharide were observed through nuclear magnetic resonance. The crystal structure of the HL-tetrasaccharide complex revealed carbohydrate chain packing into the A, B, C, and D binding sites of HL, which primarily occurred through residue-specific, direct or water-mediated hydrogen bonds and hydrophobic contacts. Overall, these results support a crucial role of the Glu35/Asp53/Trp63/Asp102 residues in HL binding to the tetrasaccharide. These observations suggest an unknown glycan-guided mechanism that underlies recognition of the bacterial cell wall by lysozyme and may complement the HL immune defense function.

We have discovered a well-defined extended conformation of double-stranded DNA, which we call Σ-DNA, using laser-tweezers force-spectroscopy experiments. At a transition force corresponding to free energy change ΔG = 1·57 ± 0·12 kcal (mol base pair)−1 60 or 122 base-pair long synthetic GC-rich sequences, when pulled by the 3′−3′ strands, undergo a sharp transition to the 1·52 ± 0·04 times longer Σ-DNA. Intriguingly, the same degree of extension is also found in DNA complexes with recombinase proteins, such as bacterial RecA and eukaryotic Rad51. Despite vital importance to all biological organisms for survival, genome maintenance and evolution, the recombination reaction is not yet understood at atomic level. We here propose that the structural distortion represented by Σ-DNA, which is thus physically inherent to the nucleic acid, is related to how recombination proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that a homogeneously stretched DNA undergoes a ‘disproportionation’ into an inhomogeneous Σ-form consisting of triplets of locally B-like perpendicularly stacked bases. This structure may ensure improved fidelity of base-pair recognition and promote rejection in case of mismatch during homologous recombination reaction. Because a triplet is the length of a gene codon, we speculate that the structural physics of nucleic acids may have biased the evolution of recombinase proteins to exploit triplet base stacks and also the genetic code.

Ahmed and I were close - at least that is what I always felt - he called me his big brother (I was one year his senior). While I was more direct about most things, even personal matters, he was generally more difficult to read and, everybody I know agrees, intrinsically quite private. However, we shared the same sense of humor and the same curious, I would say almost childlike, mindset when approaching scientific problems - though his was that of a genius. Ahmed was, despite his deeper felt reservation, very social, with an eye for everybody, whether a janitor or a Nobel laureate. To really see people is something I think I learnt very much from him and indeed find very important in life. His social ability was always combined with humor: I remember many a good laugh together. At the same time, he was reflective and a deep philosopher - often finding scholarly parallels with ancient Egyptian or Greek science….

The binding mode of thionine (3,7-diamino-5-phenothiazinium) with alternating and non-alternating DNA polynucleotides at low binding ratios was conclusively determined using linear and circular dichroism spectroscopies. The binding to [poly(dG-dC)]

Structural conversion of one given protein sequence into different amyloid states, resulting in distinct phenotypes, is one of the most intriguing phenomena of protein biology. Despite great efforts the structural origin of prion diversity remains elusive, mainly because amyloids are insoluble yet noncrystalline and therefore not easily amenable to traditional structural-biology methods. We investigate two different phenotypic prion strains, weak and strong, of yeast translation termination factor Sup35 with respect to angular orientation of tyrosines using polarized light spectroscopy. By applying a combination of alignment methods the degree of fiber orientation can be assessed, which allows a relatively accurate determination of the aromatic ring angles. Surprisingly, the strains show identical average orientations of the tyrosines, which are evenly spread through the amyloid core. Small variations between the two strains are related to the local environment of a fraction of tyrosines outside the core, potentially reflecting differences in fibril packing.

Peptide AF-16, which includes the active site of Antisecretory Factor protein, has antisecretory and anti-inflammatory properties, making it a potent drug candidate for treatment of secretory and inflammatory diseases such as diarrhea, inflammatory bowel diseases, and intracranial hypertension. Despite remarkable physiological effects and great pharmaceutical need for drug discovery, very little is yet understood about AF-16 mechanism of action. In order to address interaction mechanisms, we investigated the binding of AF-16 to sulfated glycosaminoglycan, heparin, with focus on the effect of pH and ionic strength, and studied the influence of cell-surface proteoglycans on cellular uptake efficiency. Confocal laser scanning microscopy and flow cytometry experiments on wild type and proteoglycan-deficient Chinese hamster ovary cells reveal an endocytotic nature of AF-16 cellular uptake that is, however, less efficient for the cells lacking cell-surface proteoglycans. Isothermal titration calorimetry provides quantitative thermodynamic data and evidence for that the peptide affinity to heparin increases at lower pH and ionic strength. Experimental data, supported by theoretical modeling, of peptide-glycosaminoglycan interaction indicate that it has a large electrostatic contribution, which will be enhanced in diseases accompanied by decreased pH and ionic strength. These observations show that cell-surface proteoglycans are of general and crucial importance for the antisecretory and anti-inflammatory activities of AF-16.

Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions. Electronic supplementary material The online version of this article (doi:10.1007/s00249-014-0958-9) contains supplementary material, which is available to authorized users.

Non-natural peptides with structures and functions similar to natural peptides have emerged lately in biomedical as well as nanotechnological contexts. They are interesting for pharmaceutical applications since they can adopt structures with new targeting potentials and because they are generally not prone to degradation by proteases. We report here a new set of peptidomimetics derived from -peptides, consisting of n units of a 1,5-disubstituted 1,2,3-triazole amino acid (5Tzl). The monomer was prepared using ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) chemistry using [RuCl2Cp*](x) as the catalyst, allowing for simpler purification and resulting in excellent yields. This achiral monomer was used to prepare peptide oligomers that are water soluble independent of peptide chain length. Conformational analysis and structural investigations of the oligomers were performed by 2D NOESY NMR experiments, and by quantum chemical calculations using the B97X-D functional. These data indicate that several conformations may co-exist with slight energetic differences. Together with their increased hydrophilicity, this feature of homo-5Tzl may prove essential for mimicking natural peptides composed of -amino acids, where the various secondary structures are achieved by side chain effects and not by the rigidity of the peptide backbone. The improved synthetic method allows for facile variation of the 5Tzl amino acid side chains, further increasing the versatility of these compounds.

Fibrillization of peptides leads to the formation of amyloid fibres, which, when in large aggregates, are responsible for diseases such as Alzheimer's and Parkinson's. Here, we show that amyloids have strong nonlinear optical absorption, which is not present in native non-fibrillized protein. Z-scan and pump-probe experiments indicate that insulin and lysozyme β-amyloids, as well as α-synuclein fibres, exhibit either two-photon, three-photon or higher multiphoton absorption processes, depending on the wavelength of light. We propose that the enhanced multiphoton absorption is due to a cooperative mechanism involving through-space dipolar coupling between excited states of aromatic amino acids densely packed in the fibrous structures. This finding will provide the opportunity to develop nonlinear optical techniques to detect and study amyloid structures and also suggests that new protein-based materials with sizable multiphoton absorption could be designed for specific applications in nanotechnology, photonics and optoelectronics.

Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F o F 1 ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F 1 performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

Einstein was wrong with his 1927 Solvay Conference claim that quantum mechanics is incomplete and incapable of describing diffraction of single particles. However, the Einstein-Podolsky-Rosen paradox of entangled pairs of particles remains lurking with its ‘spooky action at a distance’. In molecules quantum entanglement can be viewed as basis of both chemical bonding and excitonic states. The latter are important in many biophysical contexts and involve coupling between subsystems in which virtual excitations lead to eigenstates of the total Hamiltonian, but not for the separate subsystems. The author questions whether atomic or photonic systems may be probed to prove that particles or photons may stay entangled over large distances and display the immediate communication with each other that so concerned Einstein. A dissociating hydrogen molecule is taken as a model of a zero-spin entangled system whose angular momenta are in principle possible to probe for this purpose. In practice, however, spins randomize as a result of interactions with surrounding fields and matter. Similarly, no experiment seems yet to provide unambiguous evidence of remaining entanglement between single photons at large separations in absence of mutual interaction, or about immediate (superluminal) communication. This forces us to reflect again on what Einstein really had in mind with the paradox, viz. a probabilistic interpretation of a wave function for an ensemble of identically prepared states, rather than as a statement about single particles. Such a prepared state of many particles would lack properties of quantum entanglement that make it so special, including the uncertainty upon which safe quantum communication is assumed to rest. An example is Zewail's experiment showing visible resonance in the dissociation of a coherently vibrating ensemble of NaI molecules apparently violating the uncertainty principle. Einstein was wrong about diffracting single photons where space-like anti-bunching observations have proven recently their non-local character and how observation in one point can remotely affect the outcome in other points. By contrast, long range photon entanglement with immediate, superluminal response is still an elusive, possibly partly misunderstood issue. The author proposes that photons may entangle over large distances only if some interaction exists via fields that cannot propagate faster than the speed of light. An experiment to settle this ‘interaction hypothesis’ is suggested.

© 2016 by John Wiley & Sons, Inc. All rights reserved. This chapter considers linear dichroism (LD), magnetic circular dichroism (MCD), and Forster resonance energy transfer (FRET) and how these phenomena are related to and may report on transition moment orientations and on molecular structure. MCD is an asset in combination with LD and fluorescence anisotropy for the deciphering of transition moment directions in biophysically important chromophores, and many examples of applications are found among nucleobases. A property corresponding to the absorption anisotropy (linear dichroism) for emission is the fluorescence anisotropy. The amino acid tryptophan, with indole as its photoactive aromatic chromophore, is the most frequently used natural fluorescent probe in biophysical contexts, due to its relatively high fluorescence quantum yield and well-resolved absorption profile in the near-UV. The single-molecule fluorescence-detected linear dichroism (smFLD) method is a useful complement to smFRET since it can help to avoid misinterpretation of false smFRET signals.

Its extraordinary self-assembly property, with potential to form nonperiodic structures with unique addressability, makes DNA ideal for fabrication of advanced nanostructures. We here demonstrate the controllable tethering of a hexagonal DNA nanostructure in two distinct orientations at the lipid bilayer of a liposome functioning as a soft-matter support. With polarized light (linear dichroism) applied to the flow-aligned liposomes, we show that the construct is preferentially in a parallel alignment with the lipid surface when two anchors are attached while with one anchor only a perpendicular orientation is observed.

Thermodynamic instability is a problem when assembling and purifying complex DNA nanostructures formed by hybridization alone. To address this issue, we have used photochemical fixation and orthogonal copper-free, ring-strain-promoted, click chemistry for the synthesis of dimeric, trimeric, and oligomeric modular DNA scaffolds from cyclic, double-stranded, 80-mer DNA nanoconstructs. This particular combination of orthogonal click reactions was more effective for nanoassembly than others explored. The complex nanostructures are stable to heat and denaturation agents and can therefore be purified and characterized. They are addressable in a sequence-specific manner by triplex formation, and they can be reversibly and selectively deconstructed. Nanostructures utilizing this orthogonal, chemical fixation methodology can be used as building blocks for nanomachines and functional DNA nanoarchitectures.

B.N. was supported by grants from the King Abdullah University of Science and Technology (KAUST). Y.N. holds a KAUST postdoctoral fellowship.

Cell-penetrating peptides (CPPs) are able to traverse cellular membranes and deliver macromolecular cargo. Uptake occurs through both endocytotic and nonendocytotic pathways, but the molecular requirements for efficient internalization are not fully understood. Here we investigate how the presence of tryptophans and their position within an oligoarginine influence uptake mechanism and efficiency. Flow cytometry and confocal fluorescence imaging are used to estimate uptake efficiency, intracellular distribution and toxicity in Chinese hamster ovarian cells. Further, membrane leakage and lipid membrane affinity are investigated. The peptides contain eight arginine residues and one to four tryptophans, the tryptophans positioned either at the N-terminus, in the middle, or evenly distributed along the amino acid sequence. Our data show that the intracellular distribution varies among peptides with different tryptophan content and backbone spacing. Uptake efficiency is higher for the peptides with four tryptophans in the middle, or evenly distributed along the peptide sequence, than for the peptide with four tryptophans at the N-terminus. All peptides display low cytotoxicity except for the one with four tryptophans at the N-terminus, which was moderately toxic. This finding is consistent with their inability to induce efficient leakage of dye from lipid vesicles. All peptides have comparable affinities for lipid vesicles, showing that lipid binding is not a decisive parameter for uptake. Our results indicate that tryptophan content and backbone spacing can affect both the CPP uptake efficiency and the CPP uptake mechanism. The low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view.

The catalytic role of hydrophobic co-solutes in DNA strand exchange is demonstrated by FRET kinetics. Two mechanisms that contribute to this are base stacking destabilisation and nucleation-promoted DNA strand invasion. We propose that hydrophobic catalysis is involved in the strand-exchange activity of recombination enzymes.

We investigate how DNA interacts with drugs in humid polyvinyl alcohol (PVA) films by using a homologous set of cyanine dyes (YO(+), YO-PRO(2+), and YOYO(4+)) known to intercalate into DNA with increasing affinity with increasing charge. UV-vis spectroscopy shows that the PVA matrix destabilizes all three DNA-dye complexes compared to aqueous solution but to a lesser degree as the dye charge increases. The monovalent YO is fully dissociated from DNA within minutes, whereas the dissociation of the divalent YO-PRO takes about one hour and occurs by a two-step mechanism. The tetravalent homodimer YOYO is even less affected by the PVA environment and remains intercalated in the B-form DNA also in the PVA films. The reduced stability of the DNA-dye complexes is discussed in terms of steric and dielectric properties of the PVA matrix. After being kept in dry PVA films for 48 h the DNA-YOYO complexes can be reformed reversibly by rehumidifying the films for 30 min. The ability to store aligned and confined DNA intercalated with ligand complexes may be useful in studies on structural properties of nucleic acids.

Cell-penetrating peptides (CPPs) gain access to intracellular compartments mainly via endocytosis and have capacity to deliver macromolecular cargo into cells. Although the involvement of various endocytic routes has been described it is still unclear which interactions are involved in eliciting an uptake response and to what extent affinity for particular cell surface components may determine the efficiency of a particular CPP. Previous biophysical studies of the interaction between CPPs and either lipid vesicles or soluble sugar-mimics of cell surface proteoglycans, the two most commonly suggested CPP binding targets, have not allowed quantitative correlations to be established. We here explore the use of plasma membrane vesicles (PMVs) derived from cultured mammalian cells as cell surface models in biophysical experiments. Further, we examine the relationship between affinity for PMVs and uptake into live cells using the CPP penetratin and two analogs enriched in arginines and lysines respectively. We show, using centrifugation to sediment PMVs, that the amount of peptide in the pellet fraction correlates linearly with the degree of cell internalization and that the relative efficiency of all-arginine and all-lysine variants of penetratin can be ascribed to their respective cell surface affinities. Our data show differences between arginine- and lysine-rich variants of penetratin that has not been previously accounted for in studies using lipid vesicles. Our data also indicate greater differences in binding affinity to PMVs than to heparin, a commonly used cell surface proteoglycan mimic. Taken together, this suggests that the cell surface interactions of CPPs are dependent on several cell surface moieties and their molecular organization on the plasma membrane.

Magnetically triggered release from magnetic giant unilamellar vesicles (GUVs) loaded with Alexa fluorescent dye was studied by means of confocal laser scanning microscopy (CLSM) under a low-frequency alternating magnetic field (LF-AMP). Core/shell cobalt ferrite nanoparticles coated with rhodamine B isothiocyanate (MP@SiO2(RITC)) were prepared and adsorbed on the GUV membrane. The MP@SiO2-(RITC) location and distribution on giant lipid vesicles were determined by 3D-CLSM projections, and their effect on the release properties and GUV permeability under a LF-AMF was investigated by CLSM time-resolved experiments. We show that the mechanism of release of the fluorescent dye during the LF-AMF exposure is induced by magnetic nanoparticle energy and mechanical vibration, which promote the perturbation of the GUV membrane without its collapse.

In a majority of living organisms, F o F 1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP + P i → ATP + H 2 O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing to the complexity of this multisubunit enzyme. Based on quantum mechanical computations using recent high resolution X-ray structures, we propose that during ATP synthesis the enzyme first prepares the inorganic phosphate for the γ P-O ADP bond-forming step via a double-proton transfer. At this step, the highly conserved αS344 side chain plays a catalytic role. The reaction thereafter progresses through another transition state (TS) having a planar ion configuration to finally form ATP. These two TSs are concluded crucial for ATP synthesis. Using stepwise scans and several models of the nucleotide-bound active site, some of the most important conformational changes were traced toward direction of synthesis. Interestingly, as the active site geometry progresses toward the ATP-favoring tight binding site, at both of these TSs, a dramatic increase in barrier heights is observed for the reverse direction, i.e., hydrolysis of ATP. This change could indicate a “ratchet” mechanism for the enzyme to ensure efficacy of ATP synthesis by shifting residue conformation and thus locking access to the crucial TSs.

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

In this work we have studied the effect of a low-frequency alternating magnetic field (LF-AMF) on the permeability of magnetoliposomes, i.e. liposomes including magnetic nanoparticles within their water pool. Large unilamellar liposomes loaded with magnetic cobalt ferrite nanoparticles (CoFe2O4) have been prepared and characterized. Structural characterization of the liposomal dispersion has been performed by dynamic light scattering (DLS). The enhancement of liposome permeability upon exposure to LF-AMF has been measured as the self-quenching decrease of a fluorescent hydrophilic molecule (carboxyfluorescein, CF) entrapped in the liposome pool. Liposome leakage has been monitored as a function of field frequency, time of exposure and concentration, charge and size of the embedded nanoparticles. The results show that CF release from magnetoliposomes is strongly promoted by LF-AMF, reasonably as a consequence of nanoparticle motions in the liposome pool at the applied frequency. CF release as a function of time in magnetoliposomes unexposed to magnetic field follows Fickian diffusion, while samples exposed to LF-AMF show zero-order kinetics, consistently with an anomalous transport, due to an alteration of the bilayer permeability. These preliminary results open up new perspectives in the use of these systems as carriers in targeted and controlled release of drugs.

Based upon the structural properties of DNA duplexes and their counterion-water surrounding in solution, we have introduced here a screw model which may describe translocation of DNA duplexes through artificial nanopores of the proper diameter (where the DNA counterion–hydration shell can be intact) in a qualitatively correct way. This model represents DNA as a kind of "screw," whereas the counterion-hydration shell is a kind of "nut." Mathematical conditions for stable dynamics of the DNA screw model are investigated in detail. When an electrical potential is applied across an artificial membrane with a nanopore, the "screw" and "nut" begin to move with respect to each other, so that their mutual rotation is coupled with their mutual translation. As a result, there are peaks of electrical current connected with the mutual translocation of DNA and its counterion–hydration shell, if DNA is possessed of some non-regular base-pair sequence. The calculated peaks of current strongly resemble those observed in the pertinent experiments. An analogous model could in principle be applied to DNA translocation in natural DNA–protein complexes of biological interest, where the role of "nut" would be played by protein-tailored "channels." In such cases, the DNA screw model is capable of qualitatively explaining chemical-to-mechanical energy conversion in DNA–protein molecular machines via symmetry breaking in DNA–protein friction.

In the present study, we use the fluorescent DNA base analog tCå to investigate the thermal stability of a small DNA hexagon and the thermodynamie factors that govern the formation of such a structure. The DNA molecule is becoming increasingly popular as a material for bottom-up construction of nanostructures; however, relatively little attention has been given to the thermodynamics of such biomacromolecule-based constructs. With the goal of increasing information density and structural complexity, the size of the nanoarchitectures decreases and, more importantly, the fine structure is becoming more detailed. In this process the thermal stability and formation of unwanted byproducts will become critical features to consider in the design and assembly of such structures. Using tCå as a fluorescent probe in fluorescence monitored DNA melting allows for individually observing the denaturing of each of the six 10-mer sides in the pseudohexagonal multicomponent system. Experimental results demonstrate that the ring-opening of the cyclized hexamer is virtually exclusive to one side and that the stability of this side is increased as a result of the cyclization. Moreover, a theoretical model describing the formation and melting of the nanostructure is presented. The results show that the cyclized structure is thermodynamically favored over linear polymeric structures under the conditions and concentrations used for the self-assembly. © 2009 American Chemical Society.

DNA strand exchange is of great importance in vivo for genetic recombination and DNA repair. The detailed mechanism of strand exchange is not understood in full detail despite extensive studies. Simplistic model systems in which molecular parameters can be varied independently are therefore of interest to study. We chose the surface of a positively charged liposome as a scaffold, which we recently demonstrated to be able to catalyze the exchange of fully complementary DNA oligonucleotides. We here study how single base pair mismatches affect the rate of strand exchange on the liposome surface. Interestingly, the rate of the exchange does not simply follow the stability of the duplex in solution, as determined by melting temperatures, but also depends sensitively on the position of the mismatch. For duplexes with similar melting temperatures, the exchange is much faster for a mismatch close to the end than for a mismatch in the middle of the sequence. Our results suggest that the single strands are stabilized by the liposome surface; therefore, the duplex is fraying more and the DNA opens up in a zipperlike fashion on the surface, increasing the probability of strand exchange. We also show that the competition between greater stability (higher T m in solution) and higher concentration is important for the final composition of the duplex when a large excess of single strands is added to a complementary double-stranded DNA. Finally, the similar exchange rate constants for fully base-paired duplexes on the liposome surface when adding fully matched single strands or single strands with a mismatched base indicate that the rate is governed largely by separation of the initial duplex and not by the formation of the product duplex. © 2009 American Chemical Society.

Properties of an on-chip photovoltaic nanodevice are demonstrated. The dyes comprise green florescent proteins (GFP). Dependence of recently reported zero external potential bias (ZEPB) photocurrent (I) on temperature, power, and wavelength (λ) is shown. Correlation between UV-vis spectrum of the GFP and the ZEPB I(λ) of the device is reported. The temperature dependence suggests the ZEPB photocurrent to reflect a liquid crystal type ordering where the current declines monotonically with increasing temperature. The influence of an external bias on the photocurrent is demonstrated. The resulting light-induced current is analyzed in terms of resistive and quantum mechanical contributions.

Ruthenium complexes with dipyridophenazine (dppz) type ligands have several characteristics that make them good candidates for use as luminescence probes for hydrophobic environments. Most studies have concerned DNA intercalation, but also lipid membrane fluidity and liposome orientation have been assessed. We report here dipyridophenazine derivatives ([Ru(phen)2dppz]2+) substituted with one or two alkyl ether chains of different lengths aimed at finding the optimum substitution for a high quantum yield when bound to a phospholipid membrane bilayer. The orientation of membrane bound molecules is studied using flow linear dichroism (LD) with phospholipid vesicles as membrane models. LD, excitation anisotropy, steady state luminescence and excited-state lifetime measurements are used to quantitatively investigate the insertion and orientation of the complexes in the vesicles. All complexes are inserted with their long axis of the dppz moiety mainly parallel to the lipid chains, and the degree of orientation is comparable to that of the orientation probe retinoic acid. The ruthenium "head group" with its positive charge functions as a buoy at the water-membrane interface while the hydrophobic chain part embeds the complex down into the bilayer. The complex with two hexyl ether substituents (named D6) has the optimal chain length regarding membrane insertion and orientation, and together with the highest quantum yield, is the best luminescence membrane probe in the two series.

We scrutinize the available (seemingly disparate) theories of protein folding and propose a new concept which brings them under one roof. First, we single out dipole–dipole coupling within protein backbone as the main reason for intrinsic double-well nature of the protein potential. Then, protein folding as a whole ought to be (at least) a two-stage process, namely: (a) both amino-acid side chains and solvent enslave the dynamics of the backbone to reach the folding transition state with the help of stochastic resonance, and (b) the backbone funnels the whole protein into the global potential energy minimum by enslaving the dynamics of the amino-acid side chains plus solvent, and simultaneously arresting the stochastic resonance prerequisites to lock the protein in its folded state. The latter is accomplished owing to the concerted action of the protein compactization (enthalpic contribution) and thermal motion intensification (entropic contribution), which is, in fact, a physical hallmark of enthalpy–entropy compensation.

(Figure Presented) Gently brought into line: Functionalized carbon nanotubes (see picture, C,H black, N blue) facilitate a high degree of orientation in a weak magnetic field H → , as detected by linear dichroism spectroscopy (incident planes of light A⊥ and A∥). In addition, relaxation measurements in the magnetic field allow the length of the nanotubes to be determined. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.