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ABSTRACT: Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.
Protein and Peptide Letters 04/2012; 19(8):791-4. · 1.94 Impact Factor
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ABSTRACT: Scanning probe microscopy-based techniques can address and manipulate individual molecules. This makes it possible to use them for building nanostructures by assembling single molecules. Recently the formation of surface structures by positioning single molecules with the Atomic Force Microscope (AFM) was demonstrated on an irreversible delivery process. This inherits the drawback, that the transfer has to occur between differently functionalized surfaces and allows no proofreading of the built structures. Here we demonstrate a procedure for directed deposition of single DNA molecules, which intrinsically allows a reversible positioning. This method uses specific interactions between complementary DNA oligonucleotides for symmetric coupling of the transport molecules to the support and AFM tip, respectively. Thus, it allows for a simple "drag-and-drop" procedure, which relies on the statistical breakage of the molecular interaction under a force load. In addition, the delivery of the transport molecules was observed in real-time by single-molecule fluorescence microscopy.
Journal of Nanoscience and Nanotechnology 08/2010; 10(8):5328-32. · 1.56 Impact Factor
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ABSTRACT: Force spectroscopy allows testing the free energy landscapes of molecular interactions. Usually, the dependency of the most probable rupture force on the force rate or the shape of the rupture force histogram is fitted with different models that contain approximations and basic assumptions. We present a simple and model free approach to extract the force-dependent dissociation rates directly from the force curve data. Simulations show that the dissociation rates at any force are given directly by the ratio of the number of detected rupture events to the time this force was acting on the bond. To calculate these total times of acting forces, all force curve data points of all curves measured are taken into account, which significantly increases the amount of information which is considered for data analysis compared to other methods. Moreover, by providing force-dependent dissociation rates this method allows direct testing and validating of any energy landscape model.
Biophysical Journal 11/2009; 97(9):L19-21. · 3.65 Impact Factor
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ABSTRACT: Microbial rhodopsins are a family of seven-helical transmembrane proteins containing retinal as chromophore. Sensory rhodopsin II (SRII) triggers two very different responses upon light excitation, depending on the presence or the absence of its cognate transducer HtrII: Whereas light activation of the NpSRII/NpHtrII complex activates a signalling cascade that initiates the photophobic response, NpSRII alone acts as a proton pump. Using single-molecule force spectroscopy, we analysed the stability of NpSRII and its complex with the transducer in the dark and under illumination. By improving force spectroscopic data analysis, we were able to reveal the localisation of occurring forces within the protein chain with a resolution of about six amino acids. Distinct regions in helices G and F were affected differently, depending on the experimental conditions. The results are generally in line with previous data on the molecular stability of NpSRII. Interestingly, new interaction sites were identified upon light activation, whose functional importance is discussed in detail.
Journal of Molecular Biology 08/2009; 394(3):383-90. · 4.00 Impact Factor
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ABSTRACT: Covalent chemisorption of biomolecules to surfaces with high density and low unspecific background is prerequisite for most optical and mechanical single molecule experiments and accordingly, many recipes have been developed. However, new establishment of the surface functionalization process in the lab usually is still difficult and time consuming due to the complex procedures containing many pitfalls. Therefore, based on the known recipes, we developed and optimized a simple straight forward protocol. We demonstrated it resulting in a high density of the coupled biomolecules, homogeneous surfaces and a low unspecific background when binding nucleic acids, peptides and proteins. The protocol was optimized for borosilicate cover glasses and silicon nitride atomic force microscope cantilevers commonly used in single molecule experiments and takes advantage of commonly used chemicals. It consists of only four steps, silanol group generation, amination, grafting of poly(ethylene glycol) to the surface and biomolecule coupling. All individual steps were optimized comparing different variations partially described in the literature. Finally, a detailed description is provided which allows avoiding most sources of contamination, often being a main hurdle on the way to single molecule experiments.
Colloids and surfaces. B, Biointerfaces 03/2009; 71(2):200-7. · 2.60 Impact Factor
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ABSTRACT: We present advances in the use of single-molecule FRET measurements with flexibly linked dyes to derive full 3D structures of DNA constructs based on absolute distances. The resolution obtained by this single-molecule approach harbours the potential to study in detail also protein- or damage-induced DNA bending. If one is to generate a geometric structural model, distances between fixed positions are needed. These are usually not experimentally accessible because of unknown fluorophore-linker mobility effects that lead to a distribution of FRET efficiencies and distances. To solve this problem, we performed studies on DNA double-helices by systematically varying donor acceptor distances from 2 to 10 nm. Analysis of dye-dye quenching and fluorescence anisotropy measurements reveal slow positional and fast orientational fluorophore dynamics, that results in an isotropic average of the FRET efficiency. We use a nonlinear conversion function based on MD simulations that allows us to include this effect in the calculation of absolute FRET distances. To obtain unique structures, we performed a quantitative statistical analysis for the conformational search in full space based on triangulation, which uses the known helical nucleic acid features. Our higher accuracy allowed the detection of sequence-dependent DNA bending by 16 degrees . For DNA with bulged adenosines, we also quantified the kink angles introduced by the insertion of 1, 3 and 5 bases to be 32 degrees +/- 6 degrees , 56 degrees +/- 4 degrees and 73 +/- 2 degrees , respectively. Moreover, the rotation angles and shifts of the helices were calculated to describe the relative orientation of the two arms in detail.
Proceedings of the National Academy of Sciences 12/2008; 105(47):18337-42. · 9.68 Impact Factor
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ABSTRACT: In haloarchaea, sensory rhodopsin II (SRII) mediates a photophobic response to avoid photo-oxidative damage in bright light. Upon light activation the receptor undergoes a conformational change that activates a tightly bound transducer molecule (HtrII), which in turn by a chain of homologous reactions transmits the signal to the chemotactic eubacterial two-component system. Here, using single-molecule force spectroscopy, we localize and quantify changes to the intramolecular interactions within SRII of Natronomonas pharaonis (NpSRII) upon NpHtrII binding. Transducer binding affected the interactions at transmembrane alpha helices F and G of NpSRII to which the transducer was in contact. Remarkably, the interactions were distributed asymmetrically and significantly stabilized alpha helix G entirely but alpha helix F only at its extracellular tip. These findings provide unique insights into molecular mechanisms that "prime" the complex for signaling, and guide the receptor toward transmitting light-activated structural changes to its cognate transducer.
Structure 09/2008; 16(8):1206-13. · 6.35 Impact Factor
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ABSTRACT: A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules.
Analytical and Bioanalytical Chemistry 02/2007; 387(1):71-82. · 3.78 Impact Factor
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ABSTRACT: The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.
RNA 11/2005; 11(10):1545-54. · 5.09 Impact Factor
