Rad51 protein, a eukaryotic homologue of RecA protein, forms a filamentous complex with DNA and c... more Rad51 protein, a eukaryotic homologue of RecA protein, forms a filamentous complex with DNA and catalyzes homologous recombination. We have analyzed the structure of Xenopus Rad51 protein (XRad51.1) in solution by small-angle neutron scattering (SANS). The measurements showed that XRad51.1 forms a helical filament independently of DNA. The sizes of the cross-sectional and helical pitch of the filament could be determined, respectively, from a Guinier plot and the position of the subsidiary maximum of SANS data. We observed that the helical structure is modified by nucleotide binding as in the case of RecA. Upon ATP binding under high-salt conditions (600 mM NaCl), the helical pitch of XRad51.1 filament was increased from 8 to 10 nm and the cross-sectional diameter decreased from 7 to 6 nm. The pitch sizes of XRad51.1 are similar to, though slightly larger than, those of RecA filament under corresponding conditions. A similar helical pitch size was observed by electron microscopy for budding yeast Rad51 [Ogawa, T., et al. (1993) Science 259, 1896-1899]. In contrast to the RecA filament, the structure of XRad51.1 filament with ADP is not significantly different from that with ATP. Thus, the hydrolysis of ATP to ADP does not modify the helical filament of XRad51.1. Together with our recent observation that ADP does not weaken the XRad51.1/DNA interaction, the effect of ATP hydrolysis on XRad51.1 nucleofilament should be very different from that on RecA.
The source of the activation energy that allows cutting of DNA by restriction enzymes is unclear.... more The source of the activation energy that allows cutting of DNA by restriction enzymes is unclear. A systematic study of the cutting efficiency of the type-II restriction endonuclease EcoRI, with varying background electrolyte ion pair and buffer reported here, shows only a modest dependence of efficiency on cation type. Surprisingly, efficiency does depend strongly on the presumed indifferent anion of the background salt. What emerges is that competition between the background salt anion and the buffer anion for the enzyme and DNA surfaces is crucial. The results are unexpected and counterintuitive from the point of view of conventional electrolyte theory. However, taken together with recent developments in surface chemistry, the results do fall into place and could also suggest a novel mechanism for enzyme activity as an alternative to metal-activated hydrolysis: microscopic cavitation in a hydrophobic pocket might be the source of activation energy.
Calculation of Binding Constants. Construction of a binding isotherm is facilitated if the assump... more Calculation of Binding Constants. Construction of a binding isotherm is facilitated if the assumption that only two states exists; peptide free in solution and peptide bound to the liposomes, can be made. Using this assumption, the titration fluorescence spectra can be projected on reference spectra of free and membrane-bound peptide (Polozov, I.V. et al.,
Upon interaction with calf thymus DNA the ∆,∆-enantiomer of the semirigid binuclear ruthenium com... more Upon interaction with calf thymus DNA the ∆,∆-enantiomer of the semirigid binuclear ruthenium complex [µ-(11,11'-bidppz)(phen) 4 Ru 2 ] 4+ has previously been shown to reorganize, from an initial groove bound geometry, into an intercalative binding mode, threading one of its bulky Ru(phen) 2 moieties through the core of the DNA. We have now found that all three stereoisomers, ∆,∆, Λ,Λ, and ∆,Λ (meso), are intercalated in their final modes of binding to calf thymus DNA, poly(dA-dT) 2 , poly(dG-dC) 2 , as well as poly(dI-dC) 2 indicated by linear dichroism, circular dichroism, and luminescence. For all three stereoisomers, studied in detail with poly(dA-dT) 2 , the bridging bidppz ligand is intercalated in anti conformation, leaving one Ru(phen) 2 moiety in each groove. This final binding geometry is characterized by a distinct clockwise roll of the Ru(phen) 2 moiety in the minor groove, similar to the roll earlier observed for the dppz ligand in [Ru(phen) 2 dppz] 2+. Using the meso stereoisomer as an enantioselective probe, it is shown that the Λ moiety prefers to insert itself deeply into the minor groove while the ∆ moiety, in the major groove, is somewhat displaced from the centre of the DNA helix. The preceding, metastable bound geometries are concluded to be in the major groove for calf thymus DNA, poly(dG-dC) 2 , and poly(dI-dC) 2 , with the ∆,∆ form displaying an angle of the bidppz bridge relative the DNA helix axis of about 50°, whereas the corresponding angles for the meso-and Λ,Λ-forms in calf thymus DNA are around 65°, suggesting an orientation in the groove more parallel to the bases. By contrast, in poly(dA-dT) 2 none of the stereoisomers exhibits any distinguishable initial groove binding mode, but all seem to bind by threading intercalation directly.
pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal e... more pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA2(1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion ( approximately 60-65 degrees relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue.
ePrints Soton (University of Southampton), May 6, 2013
U ltrafast laser modification of transparent materials is an important technique enabling product... more U ltrafast laser modification of transparent materials is an important technique enabling production of 3D photonic structures whose practical applications are rapidly widening. The physics behind laser-induced modifications is extremely rich and involves a variety of consecutive processes initiated by radiation absorption during the laser pulse and extending to millisecond timescales when the final structure becomes "frozen" in the material matrix. The quality of the final structures depends of the synergetic action of excitation of confined electron plasma, its relaxation with drawing matter into different thermodynamic states from soft heating to extreme conditions, generation of GPa pressures resulting in shock-induced material deformations, reforming of covalent bonds upon photo-excitation of the material network. In this report, we will review the physical processes responsible for various forms of laser-induced modification in wide-bandgap materials, including volume nanograting formation. We will present the modeling results obtained on the basis of the Maxwell's equations supplemented with equations describing the dynamics of the laser-induced electron plasma on the example of silica glass for typical experimental conditions. The temperature and associated stress levels are mapped in the laser energy absorption zone which may be foreseen at the end of electron-glass matrix relaxation, enabling to make conclusions on the routes of glass modification. Finally, the energy balance is considered, matching the free electron density and temperature with several threshold values (melting, plastic deformation, material failure with void formation, sublimation).
Systematical differential calorimetry experiments on DNA oligomers with different lengths and pla... more Systematical differential calorimetry experiments on DNA oligomers with different lengths and placed in water solutions with various added salt concentrations may, in principle, unravel important information about the structure and dynamics of the DNA and their water-counterion surrounding. With this in mind, to reinterpret the most recent results of calorimetric experiments on DNA oligomers of such a kind, the recent enthalpy-entropy compensation theory has been used. It is demonstrated that the application of the latter could enable direct estimation of thermodynamic parameters of the microphase transitions connected to the changes in DNA dynamical regimes versus the length of the biopolymers and the ionic strengths of their water solutions, and this calls for much more systematical experimental and theoretical studies in this field.
Biochimica Et Biophysica Acta - Biomembranes, 2011
In the search for methods to study structure and function of membrane-associated proteins and pep... more In the search for methods to study structure and function of membrane-associated proteins and peptides flow linear dichroism, LD, spectroscopy has emerged as a promising technique. Using shear-aligned lipid vesicles, conformations and binding geometries of membrane-bound bio-macromolecules can be assessed. Here we investigate anchoring properties and specific orientations of tryptophan relative to the peptide backbone and to the membrane normal for the model peptides gramicidin and melittin. We have monitored the conformational change associated with the refolding of non-channel gramicidin into its channel form, and quantitatively determined the average orientations of its tryptophan transition moments, suggesting that these residues adopt a well-defined orientation at the membrane interface. An important conclusion regards the structural variation of gramicidin between these two distinct transmembrane forms. Whilst circular dichroism (CD) spectra, as has been reported before, vary strongly between the two forms suggesting their structures might be quite different, the LD results clearly evidence both the peptide backbone orientation and tryptophan side-chain positioning to be very similar. The latter are oriented in accord with what is expected from their role to anchor peptide termini to the membrane surface. The variations in CD could be due to, the in LD observed, minor shifts in mutual orientation and distance between neighbouring tryptophans sensitively determining their exciton interactions. Our data dispute that the nonchannel form of membrane-bound gramicidin would be any of the intertwined forms often observed in crystal as the positioning of tryptophans along the peptide axis would not be compatible with the strong interfacial positioning observed here. The general role of tryptophans as interfacial anchors is further assessed for melittin whose conformation shows considerable angular spread, consistent with a carpet model of its mechanism for induced membrane leakage, and a predominantly surface-aligned membrane orientation governed by amphipathic interactions.
Biochimica Et Biophysica Acta - Biomembranes, Jul 1, 2011
Cell-penetrating peptides (CPPs) gain access to intracellular compartments mainly via endocytosis... more 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.
We scrutinize the available (seemingly disparate) theories of protein folding and propose a new c... more 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.
Biochimica Et Biophysica Acta - Biomembranes, Dec 1, 2010
The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 r... more The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 remain illdefined. Here, we have investigated the membrane disruption capacity of LL-37 using large unilamellar vesicles (LUVs) composed of varying mixtures of POPC, POPG and cholesterol to mimic target and host membranes respectively. We show that LL-37 is unable to induce leakage of entrapped calcein from zwitterionic POPC LUVs, whereas leakage from LUVs partially composed of POPG is fast and efficient. In accordance with typical antimicrobial peptide behavior, cholesterol diminished LL-37 induced leakage. By using linear dichroism and flow oriented LUVs, we found that LL-37 orients with the axis of its induced αhelix parallel to the membrane surface in POPC:POPG (7:3) LUVs. In the same system, we also observed a time-dependent increase of the parallel α-helix LD signal on timescales corresponding to the leakage kinetics. The increased LD may be connected to a peptide translocation step, giving rise to mass balance across the membrane. This could end the leakage process before it is complete, similar to what we have observed. Confocal microscopy studies of eukaryotic cells show that LL-37 is able to mediate the cell delivery of non-covalently linked fluorescent oligonucleotides, in agreement with earlier studies on delivery of plasmid DNA (Sandgren et al., J. Biol. Chem. 279 (2004) 17951). These observations highlight the potential dual functions of LL-37 as an antimicrobial agent against bacterial target cells and a cell-penetrating peptide that can deliver nucleic acids into the host cells.
2 , postulates that the strand passage mechanism of type II topoisomerases is governed by a kinet... more 2 , postulates that the strand passage mechanism of type II topoisomerases is governed by a kinetic proofreading process. In practice, this model suggests that type II topoisomerases require two collisions between the T-and G-segments prior to strand passage. The first collision of a T-segment with the enzyme-bound G-segment transiently activates the enzyme and the second collision of the T-segment with the activated enzyme results in strand passage. The model predicts that the strand passage probability scales as the square of the collision rate, which has not been tested. We directly tested this prediction of the kinetic proofreading model using a single-crossing DNA unlinking assay. We measured the rate that topoisomerase IV, a bacterial type II topoisomerase, unlinked DNA as a function of the strand collision probability obtained from Monte Carlo simulations of the DNA crossings. The unlinking rate was linearly related to the collision probability, which is inconsistent with the kinetic proofreading model.
Biochimica Et Biophysica Acta - Biomembranes, Nov 1, 2012
Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating pepti... more Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved internalization, we used flow cytometry to examine uptake in relation to cell surface binding for penetratin and two arginine/lysine substituted variants (PenArg and PenLys) in wildtype CHO-K1 and PG-deficient A745 cells. All peptides were more efficiently internalized into CHO-K1 than into A745, but their cell surface binding was independent of cell type. Thus, PGs promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Uptake of each peptide was linearly dependent on its cell surface binding, and affinity is thus important for efficiency. However, the gradients of these linear dependencies varied significantly. Thus each peptide's ability to stimulate uptake once bound to the cell surface is reliant on formation of specific uptake-promoting interactions. Heparin affinity chromatography and clustering experiments showed that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts through electrostatic attraction. This may have implications for the molecular mechanisms behind arginine-specific uptake stimulation as penetratin and PenArg are more efficiently internalized than PenLys upon interaction with PGs. However, PenArg is also least affected by removal of PGs. This indicates that an increased arginine content not only improve PG-dependent uptake but also that PenArg is more adaptable as it can use several portals of entry into the cell.
Electrostatic shielding and steric blocking by histones are two significant factors that particip... more Electrostatic shielding and steric blocking by histones are two significant factors that participate in the control of the local rates of transcription in chromatin. As a simple model system to determine how the degree of DNA condensation affects enzyme accessibility and gene expression, we have used generation 5 polyamidoamine (G5 PAMAM) cationic dendrimer particles (size 5.4 nm) as a synthetic histone model together with an in vitro transcription assay. The degree of compaction, conformation, and binding availability of the dendrimer-DNA complexes is characterized by linear and circular dichroism, dynamic light scattering, and competitive binding of ethidium. Using ultracentrifugation we are able to show explicitly, for the first time, that dendrimer particles bind to DNA in a highly cooperative manner, and that the dendrimer-induced condensation of the DNA strongly attenuates transcription. Two fractions with different properties can be identified: a low-density fraction which behaves very similar to uncondensed DNA and a high-density fraction which is condensed to a high extent and where binding availability and transcription are strongly reduced. Circular dichroism gives clues to the structure of the condensed DNA indicating long-range order between the helices such as in polymer-salt-induced cholesteric liquid crystalline domains, one possible shape being a toroidal structure. On the basis of the experimental data, we propose a model for the self-assembly of the dendrimer-DNA system.
There is a great need for development of independent methods to study the structure and function ... more There is a great need for development of independent methods to study the structure and function of membraneassociated proteins and peptides. Polarized light spectroscopy (linear dichroism, LD) using shear-aligned lipid vesicles as model membranes has emerged as a promising tool for the characterization of the binding geometry of membrane-bound biomolecules. Here we explore the potential of retinoic acid, retinol, and retinal to function as probes of the macroscopic alignment of shear-deformed 100 nm liposomes. The retinoids display negative LD, proving their preferred alignment perpendicular to the membrane surface. The magnitude of the LD indicates the order retinoic acid > retinol > retinal regarding the degree of orientation in all tested lipid vesicle types. It is concluded that mainly nonspecific electrostatic interactions govern the apparent orientation of the retinoids within the bilayer. We propose a simple model for how the effective orientation may be related to the polarity of the end groups of the retinoid probes, their insertion depths, and their angular distribution of configurations around the membrane normal. Further, we provide evidence that the retinoids can sense subtle structural differences due to variations in membrane composition and we explore the pH sensitivity of retinoic acid, which manifests in variations in absorption maximum wavelength in membranes of varying surface charge. Based on LD measurements on cholesterol-containing liposomes, the influence of membrane constituents on bending rigidity and vesicle deformation is considered in relation to the macroscopic alignment, as well as to lipid chain order on the microscopic scale.
Biochimica Et Biophysica Acta - Biomembranes, Jun 1, 2007
Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that ... more Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that transport of charge across a membrane should be formally associated with an extremely high Born energy barrier. We studied partitioning of several derivatives of the CPP penetratin in a wateroctanol two-phase system in presence of natural phospholipids to explore if solvation by ion-pairing to hydrophobic counter-ions may serve as a mechanism for cell internalisation. We demonstrate that anionic lipids can aid peptide partitioning into octanol. Particularly efficient partitioning into octanol is observed with an arginine-rich penetratin compared to a lysine-rich derivative. Substituting tryptophans for phenylalanines results in poor partitioning into octanol, due to decreased overall peptide hydrophobicity. Partitioning into octanol is dependent of phospholipid type and the peptides induced structural changes in the lipid assemblies found in octanol. Attachment of carboxyfluorescein as a model cargo was found to enhance peptide partitioning into octanol. We discuss our results with respect to theoretical electrostatic energies, empirical hydrophobicity scales and in terms of implications for CPP uptake mechanisms. An important improvement of the theoretical transfer energies is obtained when, instead of singular ions, the insertion of ion-paired dipolar species is considered.
In various chemical systems enthalpy-entropy compensation (EEC) is a well-known rule of behavior,... more In various chemical systems enthalpy-entropy compensation (EEC) is a well-known rule of behavior, although the physical roots of it are still not completely understood. It has been frequently questioned whether EEC is a truly physical phenomenon or a coincidence due to trivial mathematical connections between statistical-mechanical parameters or even simpler, a phantom effect resulting from the misinterpretation of experimental data. Here, we review EEC from a new standpoint and conclude that it may be rationalized in terms of hidden but physically real factors implying a Carnot-cycle model in which a micro-phase transition (MPT) plays a crucial role. Examples of such MPTs underlying physically valid EEC should be typically cooperative processes in supramolecular aggregates, like changes of structured water at hydrophobic surfaces, conformational transitions upon ligand-biopolymer binding, and so forth. The MPT notion could help rationalize the occurrence of EEC in connection with hydration and folding of proteins, functioning of molecular motors, and similar phenomena.
In this work we demonstrate how polarized light absorption spectroscopy (linear dichroism (LD)) a... more In this work we demonstrate how polarized light absorption spectroscopy (linear dichroism (LD)) analysis of the peptide ultraviolet-visible spectrum of a membrane-associated protein (cytochrome (cyt) c) allows orientation and structure to be assessed with quite high accuracy in a native membrane environment that can be systematically varied with respect to lipid composition. Cyt c binds strongly to negatively charged lipid bilayers with a distinct orientation in which its a-helical segments are on average parallel to the membrane surface. Further information is provided by the LD of the p-p* transitions of the heme porphyrin and transitions of aromatic residues, mainly a single tryptophan. A good correlation with NMR data was found, and combining NMR structural data with LD angular data allowed the whole protein to be docked to the lipid membrane. When the redox state of cyt c was changed, distinct variations in the LD spectrum of the heme Soret band were seen corresponding to changes in electronic transition energies; however, no significant change in the overall protein orientation or structure was observed. Cyt c is known to interact in a specific manner with the doubly negatively charged lipid cardiolipin, and incorporation of this lipid into the membrane at physiologically relevant levels was indeed found to affect the protein orientation and its a-helical content. The detail in which cyt c binding is described in this study shows the potential of LD spectroscopy using shear-deformed lipid vesicles as a new methodology for exploring membrane protein structure and orientation.
This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin prote... more This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.
Rad51 protein, a eukaryotic homologue of RecA protein, forms a filamentous complex with DNA and c... more Rad51 protein, a eukaryotic homologue of RecA protein, forms a filamentous complex with DNA and catalyzes homologous recombination. We have analyzed the structure of Xenopus Rad51 protein (XRad51.1) in solution by small-angle neutron scattering (SANS). The measurements showed that XRad51.1 forms a helical filament independently of DNA. The sizes of the cross-sectional and helical pitch of the filament could be determined, respectively, from a Guinier plot and the position of the subsidiary maximum of SANS data. We observed that the helical structure is modified by nucleotide binding as in the case of RecA. Upon ATP binding under high-salt conditions (600 mM NaCl), the helical pitch of XRad51.1 filament was increased from 8 to 10 nm and the cross-sectional diameter decreased from 7 to 6 nm. The pitch sizes of XRad51.1 are similar to, though slightly larger than, those of RecA filament under corresponding conditions. A similar helical pitch size was observed by electron microscopy for budding yeast Rad51 [Ogawa, T., et al. (1993) Science 259, 1896-1899]. In contrast to the RecA filament, the structure of XRad51.1 filament with ADP is not significantly different from that with ATP. Thus, the hydrolysis of ATP to ADP does not modify the helical filament of XRad51.1. Together with our recent observation that ADP does not weaken the XRad51.1/DNA interaction, the effect of ATP hydrolysis on XRad51.1 nucleofilament should be very different from that on RecA.
The source of the activation energy that allows cutting of DNA by restriction enzymes is unclear.... more The source of the activation energy that allows cutting of DNA by restriction enzymes is unclear. A systematic study of the cutting efficiency of the type-II restriction endonuclease EcoRI, with varying background electrolyte ion pair and buffer reported here, shows only a modest dependence of efficiency on cation type. Surprisingly, efficiency does depend strongly on the presumed indifferent anion of the background salt. What emerges is that competition between the background salt anion and the buffer anion for the enzyme and DNA surfaces is crucial. The results are unexpected and counterintuitive from the point of view of conventional electrolyte theory. However, taken together with recent developments in surface chemistry, the results do fall into place and could also suggest a novel mechanism for enzyme activity as an alternative to metal-activated hydrolysis: microscopic cavitation in a hydrophobic pocket might be the source of activation energy.
Calculation of Binding Constants. Construction of a binding isotherm is facilitated if the assump... more Calculation of Binding Constants. Construction of a binding isotherm is facilitated if the assumption that only two states exists; peptide free in solution and peptide bound to the liposomes, can be made. Using this assumption, the titration fluorescence spectra can be projected on reference spectra of free and membrane-bound peptide (Polozov, I.V. et al.,
Upon interaction with calf thymus DNA the ∆,∆-enantiomer of the semirigid binuclear ruthenium com... more Upon interaction with calf thymus DNA the ∆,∆-enantiomer of the semirigid binuclear ruthenium complex [µ-(11,11'-bidppz)(phen) 4 Ru 2 ] 4+ has previously been shown to reorganize, from an initial groove bound geometry, into an intercalative binding mode, threading one of its bulky Ru(phen) 2 moieties through the core of the DNA. We have now found that all three stereoisomers, ∆,∆, Λ,Λ, and ∆,Λ (meso), are intercalated in their final modes of binding to calf thymus DNA, poly(dA-dT) 2 , poly(dG-dC) 2 , as well as poly(dI-dC) 2 indicated by linear dichroism, circular dichroism, and luminescence. For all three stereoisomers, studied in detail with poly(dA-dT) 2 , the bridging bidppz ligand is intercalated in anti conformation, leaving one Ru(phen) 2 moiety in each groove. This final binding geometry is characterized by a distinct clockwise roll of the Ru(phen) 2 moiety in the minor groove, similar to the roll earlier observed for the dppz ligand in [Ru(phen) 2 dppz] 2+. Using the meso stereoisomer as an enantioselective probe, it is shown that the Λ moiety prefers to insert itself deeply into the minor groove while the ∆ moiety, in the major groove, is somewhat displaced from the centre of the DNA helix. The preceding, metastable bound geometries are concluded to be in the major groove for calf thymus DNA, poly(dG-dC) 2 , and poly(dI-dC) 2 , with the ∆,∆ form displaying an angle of the bidppz bridge relative the DNA helix axis of about 50°, whereas the corresponding angles for the meso-and Λ,Λ-forms in calf thymus DNA are around 65°, suggesting an orientation in the groove more parallel to the bases. By contrast, in poly(dA-dT) 2 none of the stereoisomers exhibits any distinguishable initial groove binding mode, but all seem to bind by threading intercalation directly.
pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal e... more pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA2(1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion ( approximately 60-65 degrees relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue.
ePrints Soton (University of Southampton), May 6, 2013
U ltrafast laser modification of transparent materials is an important technique enabling product... more U ltrafast laser modification of transparent materials is an important technique enabling production of 3D photonic structures whose practical applications are rapidly widening. The physics behind laser-induced modifications is extremely rich and involves a variety of consecutive processes initiated by radiation absorption during the laser pulse and extending to millisecond timescales when the final structure becomes "frozen" in the material matrix. The quality of the final structures depends of the synergetic action of excitation of confined electron plasma, its relaxation with drawing matter into different thermodynamic states from soft heating to extreme conditions, generation of GPa pressures resulting in shock-induced material deformations, reforming of covalent bonds upon photo-excitation of the material network. In this report, we will review the physical processes responsible for various forms of laser-induced modification in wide-bandgap materials, including volume nanograting formation. We will present the modeling results obtained on the basis of the Maxwell's equations supplemented with equations describing the dynamics of the laser-induced electron plasma on the example of silica glass for typical experimental conditions. The temperature and associated stress levels are mapped in the laser energy absorption zone which may be foreseen at the end of electron-glass matrix relaxation, enabling to make conclusions on the routes of glass modification. Finally, the energy balance is considered, matching the free electron density and temperature with several threshold values (melting, plastic deformation, material failure with void formation, sublimation).
Systematical differential calorimetry experiments on DNA oligomers with different lengths and pla... more Systematical differential calorimetry experiments on DNA oligomers with different lengths and placed in water solutions with various added salt concentrations may, in principle, unravel important information about the structure and dynamics of the DNA and their water-counterion surrounding. With this in mind, to reinterpret the most recent results of calorimetric experiments on DNA oligomers of such a kind, the recent enthalpy-entropy compensation theory has been used. It is demonstrated that the application of the latter could enable direct estimation of thermodynamic parameters of the microphase transitions connected to the changes in DNA dynamical regimes versus the length of the biopolymers and the ionic strengths of their water solutions, and this calls for much more systematical experimental and theoretical studies in this field.
Biochimica Et Biophysica Acta - Biomembranes, 2011
In the search for methods to study structure and function of membrane-associated proteins and pep... more In the search for methods to study structure and function of membrane-associated proteins and peptides flow linear dichroism, LD, spectroscopy has emerged as a promising technique. Using shear-aligned lipid vesicles, conformations and binding geometries of membrane-bound bio-macromolecules can be assessed. Here we investigate anchoring properties and specific orientations of tryptophan relative to the peptide backbone and to the membrane normal for the model peptides gramicidin and melittin. We have monitored the conformational change associated with the refolding of non-channel gramicidin into its channel form, and quantitatively determined the average orientations of its tryptophan transition moments, suggesting that these residues adopt a well-defined orientation at the membrane interface. An important conclusion regards the structural variation of gramicidin between these two distinct transmembrane forms. Whilst circular dichroism (CD) spectra, as has been reported before, vary strongly between the two forms suggesting their structures might be quite different, the LD results clearly evidence both the peptide backbone orientation and tryptophan side-chain positioning to be very similar. The latter are oriented in accord with what is expected from their role to anchor peptide termini to the membrane surface. The variations in CD could be due to, the in LD observed, minor shifts in mutual orientation and distance between neighbouring tryptophans sensitively determining their exciton interactions. Our data dispute that the nonchannel form of membrane-bound gramicidin would be any of the intertwined forms often observed in crystal as the positioning of tryptophans along the peptide axis would not be compatible with the strong interfacial positioning observed here. The general role of tryptophans as interfacial anchors is further assessed for melittin whose conformation shows considerable angular spread, consistent with a carpet model of its mechanism for induced membrane leakage, and a predominantly surface-aligned membrane orientation governed by amphipathic interactions.
Biochimica Et Biophysica Acta - Biomembranes, Jul 1, 2011
Cell-penetrating peptides (CPPs) gain access to intracellular compartments mainly via endocytosis... more 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.
We scrutinize the available (seemingly disparate) theories of protein folding and propose a new c... more 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.
Biochimica Et Biophysica Acta - Biomembranes, Dec 1, 2010
The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 r... more The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 remain illdefined. Here, we have investigated the membrane disruption capacity of LL-37 using large unilamellar vesicles (LUVs) composed of varying mixtures of POPC, POPG and cholesterol to mimic target and host membranes respectively. We show that LL-37 is unable to induce leakage of entrapped calcein from zwitterionic POPC LUVs, whereas leakage from LUVs partially composed of POPG is fast and efficient. In accordance with typical antimicrobial peptide behavior, cholesterol diminished LL-37 induced leakage. By using linear dichroism and flow oriented LUVs, we found that LL-37 orients with the axis of its induced αhelix parallel to the membrane surface in POPC:POPG (7:3) LUVs. In the same system, we also observed a time-dependent increase of the parallel α-helix LD signal on timescales corresponding to the leakage kinetics. The increased LD may be connected to a peptide translocation step, giving rise to mass balance across the membrane. This could end the leakage process before it is complete, similar to what we have observed. Confocal microscopy studies of eukaryotic cells show that LL-37 is able to mediate the cell delivery of non-covalently linked fluorescent oligonucleotides, in agreement with earlier studies on delivery of plasmid DNA (Sandgren et al., J. Biol. Chem. 279 (2004) 17951). These observations highlight the potential dual functions of LL-37 as an antimicrobial agent against bacterial target cells and a cell-penetrating peptide that can deliver nucleic acids into the host cells.
2 , postulates that the strand passage mechanism of type II topoisomerases is governed by a kinet... more 2 , postulates that the strand passage mechanism of type II topoisomerases is governed by a kinetic proofreading process. In practice, this model suggests that type II topoisomerases require two collisions between the T-and G-segments prior to strand passage. The first collision of a T-segment with the enzyme-bound G-segment transiently activates the enzyme and the second collision of the T-segment with the activated enzyme results in strand passage. The model predicts that the strand passage probability scales as the square of the collision rate, which has not been tested. We directly tested this prediction of the kinetic proofreading model using a single-crossing DNA unlinking assay. We measured the rate that topoisomerase IV, a bacterial type II topoisomerase, unlinked DNA as a function of the strand collision probability obtained from Monte Carlo simulations of the DNA crossings. The unlinking rate was linearly related to the collision probability, which is inconsistent with the kinetic proofreading model.
Biochimica Et Biophysica Acta - Biomembranes, Nov 1, 2012
Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating pepti... more Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved internalization, we used flow cytometry to examine uptake in relation to cell surface binding for penetratin and two arginine/lysine substituted variants (PenArg and PenLys) in wildtype CHO-K1 and PG-deficient A745 cells. All peptides were more efficiently internalized into CHO-K1 than into A745, but their cell surface binding was independent of cell type. Thus, PGs promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Uptake of each peptide was linearly dependent on its cell surface binding, and affinity is thus important for efficiency. However, the gradients of these linear dependencies varied significantly. Thus each peptide's ability to stimulate uptake once bound to the cell surface is reliant on formation of specific uptake-promoting interactions. Heparin affinity chromatography and clustering experiments showed that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts through electrostatic attraction. This may have implications for the molecular mechanisms behind arginine-specific uptake stimulation as penetratin and PenArg are more efficiently internalized than PenLys upon interaction with PGs. However, PenArg is also least affected by removal of PGs. This indicates that an increased arginine content not only improve PG-dependent uptake but also that PenArg is more adaptable as it can use several portals of entry into the cell.
Electrostatic shielding and steric blocking by histones are two significant factors that particip... more Electrostatic shielding and steric blocking by histones are two significant factors that participate in the control of the local rates of transcription in chromatin. As a simple model system to determine how the degree of DNA condensation affects enzyme accessibility and gene expression, we have used generation 5 polyamidoamine (G5 PAMAM) cationic dendrimer particles (size 5.4 nm) as a synthetic histone model together with an in vitro transcription assay. The degree of compaction, conformation, and binding availability of the dendrimer-DNA complexes is characterized by linear and circular dichroism, dynamic light scattering, and competitive binding of ethidium. Using ultracentrifugation we are able to show explicitly, for the first time, that dendrimer particles bind to DNA in a highly cooperative manner, and that the dendrimer-induced condensation of the DNA strongly attenuates transcription. Two fractions with different properties can be identified: a low-density fraction which behaves very similar to uncondensed DNA and a high-density fraction which is condensed to a high extent and where binding availability and transcription are strongly reduced. Circular dichroism gives clues to the structure of the condensed DNA indicating long-range order between the helices such as in polymer-salt-induced cholesteric liquid crystalline domains, one possible shape being a toroidal structure. On the basis of the experimental data, we propose a model for the self-assembly of the dendrimer-DNA system.
There is a great need for development of independent methods to study the structure and function ... more There is a great need for development of independent methods to study the structure and function of membraneassociated proteins and peptides. Polarized light spectroscopy (linear dichroism, LD) using shear-aligned lipid vesicles as model membranes has emerged as a promising tool for the characterization of the binding geometry of membrane-bound biomolecules. Here we explore the potential of retinoic acid, retinol, and retinal to function as probes of the macroscopic alignment of shear-deformed 100 nm liposomes. The retinoids display negative LD, proving their preferred alignment perpendicular to the membrane surface. The magnitude of the LD indicates the order retinoic acid > retinol > retinal regarding the degree of orientation in all tested lipid vesicle types. It is concluded that mainly nonspecific electrostatic interactions govern the apparent orientation of the retinoids within the bilayer. We propose a simple model for how the effective orientation may be related to the polarity of the end groups of the retinoid probes, their insertion depths, and their angular distribution of configurations around the membrane normal. Further, we provide evidence that the retinoids can sense subtle structural differences due to variations in membrane composition and we explore the pH sensitivity of retinoic acid, which manifests in variations in absorption maximum wavelength in membranes of varying surface charge. Based on LD measurements on cholesterol-containing liposomes, the influence of membrane constituents on bending rigidity and vesicle deformation is considered in relation to the macroscopic alignment, as well as to lipid chain order on the microscopic scale.
Biochimica Et Biophysica Acta - Biomembranes, Jun 1, 2007
Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that ... more Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that transport of charge across a membrane should be formally associated with an extremely high Born energy barrier. We studied partitioning of several derivatives of the CPP penetratin in a wateroctanol two-phase system in presence of natural phospholipids to explore if solvation by ion-pairing to hydrophobic counter-ions may serve as a mechanism for cell internalisation. We demonstrate that anionic lipids can aid peptide partitioning into octanol. Particularly efficient partitioning into octanol is observed with an arginine-rich penetratin compared to a lysine-rich derivative. Substituting tryptophans for phenylalanines results in poor partitioning into octanol, due to decreased overall peptide hydrophobicity. Partitioning into octanol is dependent of phospholipid type and the peptides induced structural changes in the lipid assemblies found in octanol. Attachment of carboxyfluorescein as a model cargo was found to enhance peptide partitioning into octanol. We discuss our results with respect to theoretical electrostatic energies, empirical hydrophobicity scales and in terms of implications for CPP uptake mechanisms. An important improvement of the theoretical transfer energies is obtained when, instead of singular ions, the insertion of ion-paired dipolar species is considered.
In various chemical systems enthalpy-entropy compensation (EEC) is a well-known rule of behavior,... more In various chemical systems enthalpy-entropy compensation (EEC) is a well-known rule of behavior, although the physical roots of it are still not completely understood. It has been frequently questioned whether EEC is a truly physical phenomenon or a coincidence due to trivial mathematical connections between statistical-mechanical parameters or even simpler, a phantom effect resulting from the misinterpretation of experimental data. Here, we review EEC from a new standpoint and conclude that it may be rationalized in terms of hidden but physically real factors implying a Carnot-cycle model in which a micro-phase transition (MPT) plays a crucial role. Examples of such MPTs underlying physically valid EEC should be typically cooperative processes in supramolecular aggregates, like changes of structured water at hydrophobic surfaces, conformational transitions upon ligand-biopolymer binding, and so forth. The MPT notion could help rationalize the occurrence of EEC in connection with hydration and folding of proteins, functioning of molecular motors, and similar phenomena.
In this work we demonstrate how polarized light absorption spectroscopy (linear dichroism (LD)) a... more In this work we demonstrate how polarized light absorption spectroscopy (linear dichroism (LD)) analysis of the peptide ultraviolet-visible spectrum of a membrane-associated protein (cytochrome (cyt) c) allows orientation and structure to be assessed with quite high accuracy in a native membrane environment that can be systematically varied with respect to lipid composition. Cyt c binds strongly to negatively charged lipid bilayers with a distinct orientation in which its a-helical segments are on average parallel to the membrane surface. Further information is provided by the LD of the p-p* transitions of the heme porphyrin and transitions of aromatic residues, mainly a single tryptophan. A good correlation with NMR data was found, and combining NMR structural data with LD angular data allowed the whole protein to be docked to the lipid membrane. When the redox state of cyt c was changed, distinct variations in the LD spectrum of the heme Soret band were seen corresponding to changes in electronic transition energies; however, no significant change in the overall protein orientation or structure was observed. Cyt c is known to interact in a specific manner with the doubly negatively charged lipid cardiolipin, and incorporation of this lipid into the membrane at physiologically relevant levels was indeed found to affect the protein orientation and its a-helical content. The detail in which cyt c binding is described in this study shows the potential of LD spectroscopy using shear-deformed lipid vesicles as a new methodology for exploring membrane protein structure and orientation.
This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin prote... more This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.
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Papers by Bengt Nordén