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PubMed2005

Effect of temperature on the nanomechanics of lipid bilayers studied by force spectroscopy.

The effect of temperature on the nanomechanical response of supported lipid bilayers has been studied by force spectroscopy with atomic force microscopy. We have experimentally proved that the force needed to puncture the lipid bilayer (Fy) is temperature dependent. The quantitative measurement of the evolution of Fy with temperature has been related to the structural changes that the surface undergoes as observed through atomic force microscopy images. These studies were carried out with three different phosphatidylcholine bilayers with different main phase transition temperature (TM), namely, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 2-dilauroyl-sn-glycero-3-phosphocholine. The solid-like phase shows a much higher Fy than the liquid-like phase, which also exhibits a jump in the force curve. Within the solid-like phase, Fy decreases as temperature is increased and suddenly drops as it approaches TM. Interestingly, a "well" in the Fy versus temperature plot occurs around TM, thus proving an "anomalous mechanical softening" around TM. Such mechanical softening has been predicted by experimental techniques and also by molecular dynamics simulations and interpreted in terms of water ordering around the phospholipid headgroups. Ion binding has been demonstrated to increase Fy, and its influence on both solid and liquid phases has also been discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2005

Single molecule recognition between cytochrome C 551 and gold-immobilized azurin by force spectroscopy.

Recent developments in single molecule force spectroscopy have allowed investigating the interaction between two redox partners, Azurin and Cytochrome C 551. Azurin has been directly chemisorbed on a gold electrode whereas cytochrome c has been linked to the atomic force microscopy tip by means of a heterobifunctional flexible cross-linker. When recording force-distance cycles, molecular recognition events could be observed, displaying unbinding forces of approximately 95 pN for an applied loading rate of 10 nN/s. The specificity of molecular recognition was confirmed by the significant decrease of unbinding probability observed in control block experiments performed adding free azurin solution in the fluid cell. In addition, the complex dissociation kinetics has been here investigated by monitoring the unbinding forces as a function of the loading rate: the thermal off-rate was estimated to be approximately 14 s(-1), much higher than values commonly estimated for complexes more stable than electron transfer complexes. Results here discussed represent the first studies on molecular recognition between two redox partners by atomic force microscopy.

Adsorption↗
PubMed2003

Cooperativity in forced unfolding of tandem spectrin repeats.

Force-driven conformational changes provide a broad basis for protein extensibility, and multidomain proteins broaden the possibilities further by allowing for a multiplicity of forcibly extended states. Red cell spectrin is prototypical in being an extensible, multidomain protein widely recognized for its contribution to erythrocyte flexibility. Atomic force microscopy has already shown that single repeats of various spectrin family proteins can be forced to unfold reversibly under extension. Recent structural data indicates, however, that the linker between triple-helical spectrin repeats is often a contiguous helix, thus raising questions as to what the linker contributes and what defines a domain mechanically. We have examined the extensible unfolding of red cell spectrins as monomeric constructs of just two, three, or four repeats from the actin-binding ends of both alpha- and beta-chains, i.e., alpha(18-21) and beta(1-4) or their subfragments. In addition to single repeat unfolding evident in sawtooth patterns peaked at relatively low forces (<50 pN at 1 nm/ms extension rates), tandem repeat unfolding is also demonstrated in ensemble-scale analyses of thousands of atomic force microscopy contacts. Evidence for extending two chains and loops is provided by force versus length scatterplots which also indicate that tandem repeat unfolding occurs at a significant frequency relative to single repeat unfolding. Cooperativity in forced unfolding of spectrin is also clearly demonstrated by a common force scale for the unfolding of both single and tandem repeats.

Elasticity↗
PubMed2003

Force measurements of the alpha5beta1 integrin-fibronectin interaction.

The interaction of the alpha(5)beta(1) integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the alpha(5)beta(1)/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between alpha(5)beta(1) and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an alpha(5)beta(1) expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the alpha(5)beta(1)/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual alpha(5)beta(1)/FN7-10 interactions. The dynamic rupture force of the alpha(5)beta(1)/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the alpha(5)beta(1)/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites.

Elasticity↗
PubMed2006

Balance of hydrophobic and electrostatic forces in the pH response of weak polyelectrolyte capsules.

A detailed study of the role of solution pH and ionic strength on the swelling behavior of capsules composed of the weak polyelectrolytes poly(4-vinylpyridine) (P4VP) and poly(methacrylic acid) (PMA) with different numbers of layers was carried out. The polyelectrolyte layers were assembled onto silicon oxide particles and multilayer formation was followed by zeta-potential measurements. Hollow capsules were investigated by scanning electron microscopy and atomic force microscopy. The pH-dependent behavior of P4VP/PMA capsules was probed in aqueous media using confocal laser scanning microscopy. All systems exhibited a pronounced swelling at the edges of stability, at pHs of 2 and 8.1. The swelling degree increased when more polymer material was adsorbed. The swollen state can be attributed to uncompensated positive and negative charges within the multilayers, and it is stabilized by counteracting hydrophobic interactions. The swelling was related to the electrostatic interactions by infrared spectroscopy and zeta-potential measurements. The stability of the capsules as well as the swelling degree at a given pH could be tuned, when the ionic strength of the medium was altered.

Biophysics↗
PubMed2005

Surface-induced hydrogelation.

The first azo dye that gels from its aqueous solution was synthesized and its moist hydrogel was investigated with electron microscopy and atomic force microscopy; the anionic azo dye in aqueous solution forms a hydrogel on cationic surfaces even at concentrations 50 times below the minimal gelation concentration.

Hydrogels↗
PubMed2006

In vitro induction of differentiation by retinoic acid in an immortalized olfactory neuronal cell line.

In this study, we used a neuronal cell line generated by transfection of rat olfactory epithelium with immortalizing recombinant oncogene E1A of adenovirus-2. The resulting 13.S.1.24 line of transformed cells expressed an antigenic phenotype of olfactory neuronal progenitors. Time-dependency assessments over 1 week of treatment indicated that apoptosis and differentiation induced by retinoic acid (RA) were concomitant. Indeed, RA altered the cell proliferation rate, but it also stimulated differentiation of surviving 13.S.1.24 cells into bipolar olfactory marker protein-immunoreactive neurons. To characterize the nature of the cells we used immunocytochemistry, optical imaging, scanning electron microscopy and atomic force microscopy.

Adenylyl Cyclases↗
PubMed2000

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates.

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

Amino Acid Substitution↗
PubMed2004

Study in mono and bilayers of the interaction of hepatitis G virus (GBV-C/HGV) synthetic antigen E2(99-118) with cell membrane phospholipids.

The interaction of the hepatitis G synthetic peptide E2(99-118) with cell membrane phospholipids of different characteristics such as dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) was studied by Langmuir isotherms. Epifluorescence microscopy and Atomic force microscopy (AFM) was also used to study interactions with DPPC. Compression isotherms of DPPC/E2(99-118) and DPPG/E2(99-118) mixed monolayers showed negative deviation from ideallity consistent with the existence of attractive interactions. The incorporation of the peptide in DPPC monolayer was also confirmed in epifluorescence microscopy and AFM studies. The peptide retarded the formation of DPPC domains and did not let the phospholipid get organized. No important differences in the interactions with DPPC (neutral) or DPPG (anionic) were found, thus suggesting that electrostatics forces do not have a predominant influence in these interactions.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2006

Determination of molecular size and shape of hyperbranched polysaccharide in solution.

The chemical structure of a water-soluble polysaccharide, coded as TM3b, extracted from sclerotia of Pleurotus tuber-rigium was analyzed to be a hyperbranched beta-D-glucan with beta-(1-->6), beta-(1-->4), and beta-(1-->3)-linked residues, with degree of branching (DB) of 57.6%. The results from size-exclusion chromatography combined with laser light scattering (SEC-LLS) revealed that the hyperbranched polysaccharide easily aggregated in 0.15 M aqueous NaCl, whereas it dispersed as individual chains in DMSO. The weight-average molecular weight (M(w)), radius of gyration, intrinsic viscosity, and chain density of TM3b in DMSO and in 0.15 M aqueous NaCl were measured with SEC-LLS, LLS, and viscometry. The results indicated that single chains and aggregates with aggregation number of 12 coexisted in the aqueous solution, whereas individual molecules of TM3b occurred in DMSO. In view of the molecular parameters, the aggregates in aqueous solution exhibited more compact chain structure than the individual molecules in DMSO. Furthermore, transmission electron microscopy and atomic force microscopy showed that all of the aggregates and individual molecules exhibited spherical particles in the solutions. This work provided the valuable information of chain conformation and molecular morphology of the hyperbranched polysaccharide in different solvents.

Animals↗
PubMed2000

Dose-dependent modulation of octacalcium phosphate crystal habit by amelogenins.

In vitro studies on interactions between amelogenins and calcium phosphate crystals are critical for elucidating biomineralization mechanisms of tooth enamel. This work was aimed at investigating the effects of native porcine amelogenins on octacalcium phosphate (OCP) crystal growth in a gelatin gel. We prepared OCP mineral discs by circulating calcium and phosphate solutions on the opposite ends of the gels loaded with 0-2% amelogenin for one week. A dose-dependent modulation of OCP crystal habit by amelogenins was observed by scanning electron microscopy. While the incorporation of 0.125, 0.25, or 0.5% amelogenins showed no significant effect on the crystal morphology, in the presence of 1 and 2% amelogenins, the crystals were remarkably longer, having an average aspect ratio 3-5 times greater than that of those formed in the control gels. Transmission electron microscopy and atomic force microscopy suggested that amelogenin assemblies selectively blocked b-axial development, resulting in the c-axial elongation of OCP crystals.

Amelogenesis↗
PubMed2005

Perpendicular magnetization in self-assembled films of citrate-capped gamma-Fe2O3 nanocrystals on Si(100) surfaces.

gamma-Fe2O3 nanocrystals capped with citrate and octylamine have been chemically prepared. The octylamine-capped nanocrystals exhibit a tendency to form ordered lattices. Films of nanocrystals of varying thickness (454, 720, and 1400 microg/cm2 in the case of citrate-capped nanocrystals and 300 microg/cm2 in the case of octylamine-capped nanocrystals) have been prepared on Si(100) substrates by drop casting and have been characterized by X-ray diffraction, scanning electron microscopy, and atomic force microscopy. Magnetic measurements have been carried out on the films as well as on nanocrystal powders. The films of citrate-capped gamma-Fe2O3 nanocrystals exhibit enhanced perpendicular magnetization, with the anisotropy depending on the film thickness.

Amines↗
PubMed2003

Mucoadhesive interactions.

The adhesive properties of certain types of biopolymer can be used to increase the residence time of orally or nasally administered drugs. A fuller understanding of the molecular processes underpinning such 'mucoadhesive' phenomena will help in the optimal design of delivery systems. The interactions involved are, however, less well defined compared with those often encountered in protein-recognition phenomena: mucoadhesive interaction products can be very large and polydisperse, so to probe them we need to adopt a different strategy to those used by protein biochemists. Reviewed herein is some of the recent work at physiological or near-physiological solution conditions involving molecular hydrodynamics--with analytical ultracentrifugation and SEC-MALLs (size-exclusion chromatography coupled to multi-angle laser light scattering) as the cornerstones--reinforced by viscometry and the imaging probes of electron microscopy and atomic force microscopy. These clearly demonstrate the mucoadhesive properties of both an unusual cationic protein [Deacon, Davis, Waite and Harding (1998) Biochemistry 37, 14108-14112] and more significantly chitosan polysaccharides of varying degrees of charge/acetylation as a function of solution conditions, and are providing the platform for the construction of stable formulations.

Animals↗
PubMed2004

Investigation of morphological changes to Staphylococcus aureus induced by ovine-derived antimicrobial peptides using TEM and AFM.

The effect of two ovine-derived peptides on the morphology of Staphylococcus aureus NCTC 4163 cells were investigated using transmission electron microscopy and atomic force microscopy. Both techniques showed that SMAP29, an alpha-helical peptide, induced cell lysis, whereas OaBac5mini, a proline/arginine-rich peptide, caused no observable morphological changes. This is consistent with previous experimental work which indicated that SMAP29 caused cell death and induced cell lysis, whereas OaBac5mini acted by interacting with the inner cellular contents.

Amino Acid Sequence↗
PubMed1998

Immobilization strategies for biological scanning probe microscopy.

Biological atomic force microscopy (AFM) is now established as a method for studying the structure and function of biomolecular objects at the solid-liquid interface. Major progress in this field is linked to new developments in instrumentation, a better understanding of tip-sample interactions, and improved sample preparation techniques. In this review, the most common strategies for biomolecular immobilization with respect to biological AFM applications are summarized.

Microscopy, Atomic Force↗
PubMed1996

Conformational states of the nuclear pore complex induced by depletion of nuclear Ca2+ stores.

The nuclear pore complex (NPC) is essential for the transit of molecules between the cytoplasm and nucleoplasm of a cell and until recently was thought to allow intermediate-sized molecules (relative molecular mass of approximately 10,000) to diffuse freely across the nuclear envelope. However, the depletion of calcium from the nuclear envelope of Xenopus laevis oocytes was shown to regulate the passage of intermediate-sized molecules. Two distinct conformational states of the NPC were observed by field emission scanning electron microscopy and atomic force microscopy. A central plug occluded the NPC channel after nuclear calcium stores had been depleted and free diffusion of intermediate-sized molecules had been blocked. Thus, the NPC conformation appears to gate molecular movement across the nuclear envelope.

Animals↗
PubMedDate not supplied

Efficacy testing for hair care products.

For the efficacy testing of hair care products, a number of methods are available. Scanning electron microscopy and atomic force microscopy are most suitable for surface evaluation; the swelling properties in the course of a treatment are determined using a fiber-swelling analyzer, and the luster may be reliably determined by means of a multiangle goniophotometer. Further, surface tension is a suitable method to evaluate the hydrophilic/hydrophobic properties of a hair section, and is sensitive enough to indicate the individual hormone cycle.

Biomechanical Phenomena↗
PubMed2006

Self-assembled nanocages for hydrophilic guest molecules.

Reverse polymeric micelles are obtained following the association of polymeric amphiphiles in apolar media. To this date, reports of pharmaceutical applications for such micelles have been scarce, mainly because these systems have been studied in solvents that are not suitable for medical use. Here, alkylated star-shaped poly(glycerol methacrylate) polymers have been proposed in the design of oil-soluble reverse polymeric micelles. Micellar behavior was studied in various apolar solvents, including ethyl oleate, a pharmaceutically acceptable vehicle. The polymers were shown to assemble into spherical nanostructures (<40 nm) as determined by cryogenic transmission electron microscopy and atomic force microscopy studies. Interestingly, the reverse micelles were able to encapsulate various peptides/proteins (vasopressin, myoglobin, and albumin) in substantial amounts and facilitate their solubilization in oil. The nature of both the polymer used in micelle formation and the guest molecules was found to influence the ability of the micelle to interact with hydrophilic compounds.

Magnetic Resonance Spectroscopy↗