Single-molecule force spectroscopy measurements of "hydrophobic bond" between tethered hexadecane molecules

The hydrophobic effect is important for many biological and technological processes. Despite progress in theory, experimental data clarifying water structure and the interaction between hydrophobic solutes at the nanometer scale are scarce due to the very low solubility of hydrophobic species. This article describes an AFM single molecule force spectroscopy method to probe the interaction between molecules with low solubility and reports measurements of the strength and the length scale of the "hydrophobic bond" between hexadecane molecules. Hexadecane molecules are tethered by flexible poly(ethylene glycol) linkers to AFM probes and substrates, removing the aggregation state uncertainty of solution-based approaches as well as spurious surface effects. A shorter hydrophilic polymer layer is added to increase the accessibility of hydrophobic molecules for the force spectroscopy measurements. Statistical analysis of the rupture forces yields a barrier width of 0.24 nm, and a dissociation rate of 1.1 s(-1). The results of single molecule measurements are related to the theoretical predictions of the free energy of cavitation in water and to the empirical model of micellization of nonionic surfactants. It is estimated that approximately one-quarter of each molecule's surface is hydrated during forced dissociation, consistent with an extended (nonglobular) conformation of the hexadecane molecules in the dimer.     

Single-molecule study of lateral mobility of epidermal growth factor receptor 2/HER2 on activation

The transmembrane protein HER2, a member of the epidermal growth factor receptor family of tyrosine kinase, plays important roles in many fundamental cellular processes as well as the pathogenesis of many cancers. In this work, we have applied the single-molecule fluorescence microscopic method to study lateral mobility change of HER2 on activation by imaging and tracking individual GFP-tagged HER2 molecules on the membrane of living cells. The single HER2 molecules displayed different diffusion rates and modes. It was interesting to find that the mobility of HER2 increased upon stimulation by heregulin beta1, the specific ligand of HER3. The faster diffusion was related to the tyrosine phosphorylation of HER2 or EGFR. The results provided new information for the understanding of HER2 activation and molecular mechanism of signal transduction through HER2/HER3 heterodimerization.     

Dynamic force spectroscopy of the specific interaction between the PDZ domain and its recognition peptides

To characterize the molecular basis of specific interactions of PDZ proteins, dynamic force spectroscopy (DFS) for the PDZ protein Tax-interacting protein-1 (TIP-1) and its recognition peptide (PDZ-pep) derived from beta-catenin was performed using an atomic force microscope (AFM), together with measurement of thermodynamic and kinetic parameters using surface plasmon resonance (SPR). The unbinding force of this pair was measured under different conditions of AFM tip-retraction velocity. The relationship between the unbinding force and the logarithmic force-loading rate, that is, the dynamic force spectrum, exhibited two different rate regimes, for each of which the forces increased linearly with the force-loading rate. On the basis of the theoretical treatment of the Bell-Evans model, the positions of two different activation barriers in the reaction coordinate and dissociation rate constants in each barrier were evaluated from slopes and x-intercepts of the two linear regimes (first barrier: 0.04 nm and 1.10 x 10 s(-1); second barrier: 0.21 nm and 2.77 x 10(-2) s(-1), respectively). Although two-step unbinding kinetics between TIP-1 and PDZ-pep was suggested from the DFS analysis, SPR results showed single-step dissociation kinetics with a rate constant of 2.89 x 10(-1) s(-1). Different shapes of the free energy profile of the unbinding process were deduced from each result of DFS and SPR. The reason for such topographic differences in the energy landscape is discussed in relation to the differences in the pathways of forced unbinding and spontaneous dissociation.     

Single-molecule monitoring in living cells by use of fluorescence microscopy

Monitoring single molecules in living cells is becoming a powerful tool for study of the location, dynamics, and kinetics of individual biomolecules in real time. In recent decades, several optical imaging techniques, for example epi-fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, quasi-TIRFM, and single-point edge excitation subdiffraction microscopy (SPEED), have been developed, and their capability of capturing single-molecule dynamics in living cells has been demonstrated. In this review, we briefly summarize recent advances in the use of these imaging techniques for monitoring single-molecules in living cells for a better understanding of important biological processes, and discuss future developments.     

Imaging of epidermal growth factor receptor on single breast cancer cells using surface-enhanced Raman spectroscopy

Epidermal growth factor receptor (EGFR) is widely used as a biomarker for pathological grading and therapeutic targeting of human cancers. This study investigates expression, spatial distribution as well as the endocytosis of EGFR in single breast cancer cells using surface-enhanced Raman spectroscopy (SERS). By incubating anti-EGFR antibody conjugated SERS nanoprobes with an EGFR-over-expressing cancer cell line, A431, EGFR localization was measured over time and found to be located primarily at the cell surface. To further validate the constructed SERS probes, we applied this SERS probes to detect the EGFR expression on breast cancer cells (MDA-MB-435, MDA-MB-231) and their counterpart cell lines in which EGFR expression was down-regulated by breast cancer metastasis suppressor 1 (BRMS1). The results showed that SERS method not only confirms immunoblot data measuring EGFR levels, but also adds new insights regarding EGFR localization and internalization in living cells which is impossible in immunoblot method. Thus, SERS provides a powerful new tool to measure biomarkers in living cancer cells.     

Resonance light scattering spectroscopy study of interaction between gold colloid and thiamazole and its analytical application

In this paper, we used resonance light scattering (RLS) spectroscopy to study the interaction between thiol-containing pharmaceutical-thiamazole and gold colloid. At pH 5.2, the resonance light scattering spectrum of gold nanoparticles has a maximum peak at 555 nm and the RLS intensity is enhanced by trace amount of thiamazole due to the interaction between thiamazole and gold colloid. The binding of colloidal gold to thiamazole results in ligand-induced aggregation of colloidal gold, which was characterized by RLS spectrum, ultraviolet-visible (UV-Vis) spectrum, and transmission electron microscopy (TEM). Based upon the study, we proposed a highly sensitive, gold colloid-based assay using RLS spectrum to detect pharmaceuticals for the first time. The mechanism of binding interaction between Au colloid and thiamazole was also discussed.     

Resonance light scattering spectroscopy study of interaction between norfloxacin and calf thymus DNA and its analytical application

The interaction between norfloxacin and calf thymus double-stranded DNA (dsDNA) has been studied by a resonance light scattering (RLS) technique with a common spectrofluorometer. The characteristics of RLS spectra, the effective factors and optimum conditions of the reaction have been investigated. In Britton-Robinson (BR) buffer (pH 5.87), norfloxacin has a maximum peak 405.5 nm and the RLS intensity is remarkably enhanced by trace amount of calf thymus dsDNA due to the interaction between norfloxacin and dsDNA. The binding of norfloxacin to DNA forms large particles, which were characterized by RLS spectrum, scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrum, and fluorescence spectrum. Based on the enhanced RLS intensity, a novel method for sensitive determination of calf thymus dsDNA concentration ranging from 0.02 to 2.3 microg ml(-1) was developed. The determination limit (3 sigma) was 1.2 ng ml(-1). The method is simple, rapid, practical and relatively free from interference generated by coexisting substance, as well as much more sensitive than most of the reported methods. Three synthetic samples of ctDNA were determined with satisfactory results.     

Single-molecule force spectroscopy of bimolecular reactions: system homology in the mechanical activation of ligand substitution reactions

The mechanochemistry of the bimolecular nucleophilic substitution of DMSO for substituted pyridines at a square-planar pincer Pd(II) center was investigated using single-molecule force spectroscopy (SMFS). The SMFS data are interpreted in terms of the Bell-Evans model, which gives thermal off-rates for two reactions that agree well with previous, stress-free measurements. The characteristic force dependency of the rupture rate, fbeta, is effectively constant for the two reactions examined (22 +/- 2 and 24 +/- 2 pN), and the system homology in the mechanical response is consistent with expected similarities in the reaction potential energy surfaces.     

Study of the effect of atorvastatin on the interaction between ICAM-1 and CD11b by live-cell single-molecule force spectroscopy

The interaction between the cell adhesion molecule CD11b and its ligand ICAM-1 plays an important role in inflammatory responses in the disease of atherosclerosis. Atorvastatin is a commonly prescribed statin drug which has been considered as one of the most potent therapeutic agents for atherosclerosis due to its lipid-lowering effect. Recently, there is a growing body of evidence that atorvastatin has anti-inflammatory effect. We have applied the advanced method of live-cell single-molecule force spectros...     

Electromotive force study on interaction between a triblock copolymer and cationic surfactants in water

The interaction between a poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) triblock copolymer F127 and three cationic surfactants with varying alkyl chain length in water were investigated in detail by electromotive force (EMF) studies. The cationic surfactants investigated are hexadecyltrimethylammonium bromide (HTAB), myristytrimethylammonium bromide (MTAB), and lauryltrimethylammonium bromide (LTAB) which have carbon numbers of 16, 14, and 12 in the alkyl chain, respectively. Obvious binding was detected by EMF measurements between F127 and HTAB. For MTAB, the binding has also been detected with a weaker strength than that of HTAB. The LTAB/F127 mixed system exhibits the weakest binding, presumably due to the too short alkyl chain. This indicates that the binding between cationic surfactants and F127 is mainly dominated by the hydrophobic/hydrophobic interaction. In addition, the interaction also shows considerable dependence on the concentration of F127 and solution parameters such as the salinity.     

FTIR spectroscopy study of the interaction between fibre of polyamide 6 and iodine

The IR spectra of several samples of polyamide 6 fibres treated with solutions of different concentrations of iodine and potassium iodide were studied. The IR spectra of their respective iodine desorptions, obtained by means of treatment with sodium thiosulphate, were also studied. The spectral changes observed are related to variations in the polymorphic crystalline α and γ forms of the polyamide, which demonstrate the formation of a complex between the tri-iodide ion and the polymer during the iodine sorption process and a structural change during desorption.     

Single-molecule analysis of interaction between p53TAD and MDM2 using aerolysin nanopores

Protein–protein interactions (PPIs) are regarded as important, but undruggable targets. Intrinsically disordered p53 transactivation domain (p53TAD) mediates PPI with mouse double minute 2 (MDM2), which is an attractive anticancer target for therapeutic intervention. Here, using aerolysin nanopores, we probed the p53TAD peptide/MDM2 interaction and its modulation by small-molecule PPI inhibitors or p53TAD phosphorylation. Although the p53TAD peptide showed short-lived (<100 ms) translocation, the protein complex induced the characteristic extraordinarily long-lived (0.1 s ∼ tens of min) current blockage, indicating that the MDM2 recruitment by p53TAD peptide almost fully occludes the pore. Simultaneously, the protein complex formation substantially reduced the event frequency of short-lived peptide translocation. Notably, the addition of small-molecule PPI inhibitors, Nutlin-3 and AMG232, or Thr18 phosphorylation of p53TAD peptide, were able to diminish the extraordinarily long-lived events and restore the short-lived translocation of the peptide rescued from the complex. Taken together, our results elucidate a novel mechanism of single-molecule sensing for analyzing PPIs and their inhibitors using aerolysin nanopores. This novel methodology may contribute to remarkable improvements in drug discovery targeted against undruggable PPIs.

Using aerolysin nanopores, we probed protein–protein interaction (PPI) between p53TAD and MDM2 and its modulation by small-molecule PPI inhibitors and p53TAD phosphorylation.     

Early detection of apoptosis in living cells by fluorescence correlation spectroscopy

Early detection of apoptotic cells via caspase activity is demonstrated with fast response time. Fluorescence correlation spectroscopy (FCS) is used to identify the presence of a cleaved fluorogenic probe based on the fluorescence of rhodamine 110 in Jurkat cells. FCS curves are shown to be markedly different for autofluorescent (non-apoptotic) cells, whereas cells with cleaved probe showed diffusion and molecular brightness characteristic of rhodamine 110. Using FCS measurements, cells were identified as apoptotic on the basis of the presence of autocorrelated fluorescence, average molecular brightness (η), and molecular dwell time (τ _D). Apoptotic cells identified in this manner were detected as early as 45 min after induction. Unlike other methods with similar identification times, such as western blotting and electron microscopy, cells remain viable for further analysis. This multi-parameter approach is rapid, flexible, and does not require transfection of the cells prior to analysis, enabling apoptosis to be identified early in a wide variety of cell types.      

EMSA and single-molecule force spectroscopy study of interactions between Bacillus subtilis single-stranded DNA-binding protein and single-stranded DNA

In this article, interactions between Bacillus subtilis single-stranded DNA binding proteins (BsSSB) and single-stranded DNA (ssDNA) were systematically studied. The effect of different molar ratios between BsSSB and ssDNA on their binding modes was first investigated by electrophoretic mobility shift assays (EMSAs). It is found that a high molar ratio of BsSSB to ssDNA can produce BsSSB-ssDNA complexes formed in the mode of two proteins binding one 65-nt (nucleotide) ssDNA whereas a low molar ratio facilitates the formation of BsSSB-ssDNA complexes in the mode of one protein binding one 65-nt ssDNA. Furthermore, two binding modes are in dynamic equilibrium. The unbinding force of BsSSB-ssDNA complexes was measured quantitatively in solutions with different salt concentrations by using AFM-based single-molecule force spectroscopy (SMFS). Our results show that the unbinding force is about 10 pN higher at high salt concentration (0.5 M NaCl) than at low salt concentration (0.1 M NaCl) and the lifetime of BsSSB-ssDNA complexes at high salt concentration is twice as long as that at low salt concentration. These results indicate that more tightly packed BsSSB-ssDNA complexes can form at high salt (0.5 M NaCl) concentration. In addition, the results of EMSA show that ssDNA, which is bound to BsSSB, can dissociate from BsSSB in the presence of the cDNA strand, indicating the dynamic nature of BsSSB-ssDNA interactions.     

Direct measurements of the interaction between pyrene and graphite in aqueous media by single molecule force spectroscopy: understanding the pi-pi interactions

Pyrene derivatives can absorb onto the surface of carbon nanotubes and graphite particles through pi-pi interactions to functionalize these inorganic building blocks with organic surface moieties. Using single molecule force spectroscopy, we have demonstrated the first direct measurement of the interaction between pyrene and a graphite surface. In particular, we have connected a pyrene molecule onto an AFM tip via a flexible poly(ethylene glycol) (PEG) chain to ensure the formation of a molecular bridge. The pi-pi interaction between pyrene and graphite is thus indicated to be approximately 55 pN with no hysteresis between the desorption and adhesion forces.     

Measurement of the anchorage force between GPI-anchored alkaline phosphatase and supported membranes by AFM force spectroscopy

Mammalian alkaline phosphatases (AP) are glycosylphosphatidylinositol (GPI) anchored proteins that are localized on the outer layer of the plasma membrane. The GPI anchors are covalently attached to the C-termini of proteins and consist of a glycan chain bonded to phosphatidylinositol with two acyl chains anchored into the membrane bilayer. Force spectroscopy, based on atomic force microscope (AFM) technology, was used to determine the adhesion of alkaline phosphatase in the absence and presence of anchors. The GPI anchors increase markedly the adhesion frequency (i.e., the protein affinity for the membrane). An adhesion force of 350 +/- 200 pN is measured between GPI-anchored AP (AP(GPI)) and supported phospholipid bilayers of dipalmitoylphosphatidylcholine (DPPC) presenting structural defects (holes). In the absence of defects, the adhesion force (103 +/- 17 pN) and the adhesion frequency are reduced. These results indicate that AP(GPI) poorly spontaneously insert into membranes in vivo and open new perspectives for the characterization of the interactions between GPI proteins and membranes.     

Proteome alterations in human hepatoma cells transfected with antisense epidermal growth factor receptor sequence

The epidermal growth factor (EGF) is a member of the growth factor superfamily that can stimulate the proliferation of many types of cells. Overexpression of EGF receptor (EGFR) was observed in many types of cancer cells. Anti-EGFR antibodies or antisense nucleic acid sequences of EGFR can suppress the growth of hepatoma cells. In order to further investigate the proteome alterations associated with malignant growth of the human hepatoma cells and the influence of EGFR signal pathway on the cellular proteome, we have comparatively analyzed the proteomes of human hepatoma cells transfected with antisense EGFR sequence (cell strain JX-1) and its control cells (cell strain JX-0) by two-dimensional (2-D) gel electrophoresis and mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 40 protein spots showed significant expression changes in JX-1 cells compared to JX-0 cells. Three of them, including the tumor suppressor protein maspin, changed with tendency to the normal levels. Two protein spots were identified as HSP27 in the same gel, and one of them had a reduced level in JX-1 cells. The apparent alterations of HSP27 in expression level might be the results from their differential chemical modifications, suggesting the effect of dynamic post-translational modifications of proteins on the growth of hepatoma cells. Other proteins such as glutathione peroxidase (GPX-1) and 14-3-3-sigma also exhibited altered expression in JX-1 cells, and their functional implications are discussed.     

The interaction mechanisms between Saccharomyces cerevisiae and menadione and its application in toxicology study

This study describes interaction mechanism between menadione and the yeast with the aid of the catabolite assay and the culture fluorescence method, with a view towards exploitting the potential of the mediator in toxicology study. Double mediator system containing menadione and the hydrophilic mediator, ferricyanide, was employed for evaluating the toxic effects of furfural on the yeast and the results showed the well consistence between the MICREDOX method and the conventional method.