Molecular order affecting electron transport through ssDNA.

DNA is considered to be the ideal model for studies of electron transport in molecule/conductor systems due to its stability, easily controlled structure and the presumed electrical properties. Scanning tunnelling microscope (STM) studies of single-stranded DNA bound to Au (111) or Au nanodots with a thiol linker were carried out under ambient conditions. The results show that the electron transfer between the STM tip and the gold is governed by the serial resistance of the oligomer strands and a water film. Electron transfer properties also depend on the alignment of the DNA strands. Measurements show that well-ordered parallel arrangement of the molecules protruding from flat crystalline surfaces is favourable for electron transport compared with unordered arrangements of molecules on spherical nanodots. Nanodots are good candidates for effective charge production by absorption of light allowing chemical reactions to happen at the dots, which can be used for storing the light energy. Understanding electron transport through molecular structures is of crucial importance for the development of such novel photovoltaic devices.

Three-dimensional bead position histograms reveal single-molecule nanomechanics.

We describe a method to investigate the structure and elasticity of macromolecules by a combination of single molecule experiments and kinematic modeling. With a photonic force microscope, we recorded spatial position histograms of a fluctuating microsphere tethered to full-length myosin-II. Assuming only that the molecule consists of concatenated rigid segments, a model derived from robot kinematics allows us to relate these histograms to the molecule's segment lengths and bending stiffnesses. Both our calculated position distributions and the experimental data show an asymmetry characteristic of a mixed entropic-enthalpic spring. Our model that fits best to experimental line profiles has two intramolecular hinges, one at the bound head domain, and another about 50 nm down the myosin tail, with a summed bending stiffness of about 3 k(B)T/rad.

Mechanical properties of single myosin molecules probed with the photonic force microscope.

To characterize elastic properties and geometrical parameters of individual, whole myosin molecules during their interaction with actin we sparsely adsorbed myosin molecules to nanometer-sized microspheres. Thermally driven position fluctuations of these microspheres were recorded with the three-dimensional detection scheme of the photonic force microscope. Upon binding of single myosin molecules to immobilized actin filaments in the absence of ATP, these thermally driven position fluctuations of the microspheres change significantly. From three-dimensional position fluctuations stiffness and geometrical information of the tethering molecule can be derived. Axial stiffness was found to be asymmetric, approximately 0.04 pN/nm for extension, approximately 0.004 pN/nm for compression. Observed stiffness of whole myosin molecules is much less than estimated for individual myosin heads in muscle fibers or for single-molecule studies on myosin fragments. The stiffness reported here, however, is identical to stiffness found in other single-molecule studies with full-length myosin suggesting that the source of this low stiffness is located outside the myosin head domain. Analysis of geometrical properties of tethering myosin molecules by Brownian dynamics computer simulations suggests a linker length of approximately 130 nm that is divided by a free hinge located approximately 90 nm above the substrate. This pivot location coincides with myosin's hinge region. We demonstrate the general applicability of thermal fluctuation analysis to determine elastic properties and geometrical factors of individual molecules.

Microcontact printing of DNA molecules.

The controlled placement of DNA molecules onto solid surfaces is the first step in the fabrication of DNA arrays. The sequential deposition of tiny drops containing the probe DNA fragments using arrays of spotting needles or ink jet nozzles has become a standard. However, a caveat of liquid spotting is the drying of the deposited drop because this creates the typical inhomogeneities, i.e., rims around the spot. Another drawback is that each DNA array is an original and has to be fabricated individually. Microcontact printing is a versatile technique to place proteins onto different target surfaces in uniformly patterned monolayers with high lateral resolution. Here, we show for the first time that DNA can also be printed with equally high resolution in the submicrometer range using an elastomeric stamp with chemically tailored surface. Two regimes for the transfer of the molecules were observed. Finally, microcontact printing of an array of DNA probes onto a solid support and its use in a subsequent hybridization assay was demonstrated.

Calcium drives fusion of SNARE-apposed bilayers.

N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) has been proposed to play a critical role in the membrane fusion process. The SNARE complex was suggested to be the minimal fusion machinery. However, there is mounting evidence for a major role of calcium in membrane fusion. Hence, the role of calcium in SNARE-induced membrane fusion was the focus of this study. It revealed that recombinant v-SNARE and t-SNARE, reconstituted into separate liposomes, interact to bring lipid vesicles into close proximity, enabling calcium to drive fusion of opposing bilayers. Exposure to calcium triggered vesicle fusion at both, high potency and efficacy. The half-time for calcium-induced fusion of SNARE-reconstituted vesicles was determined to be approximately 10 s, which is two orders of magnitude faster than in its absence. Calcium acts downstream of SNAREs, since the presence of SNAREs in bilayers increases the potency of calcium-induced vesicle fusion, without significantly influencing its efficacy. Hence, this study suggests that in the physiological state in cells, both SNAREs and calcium operate as the minimal fusion machinery.

SNAREs in opposing bilayers interact in a circular array to form conducting pores.

The process of fusion at the nerve terminal is mediated via a specialized set of proteins in the synaptic vesicles and the presynaptic membrane. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion. The structure and arrangement of these SNAREs associated with lipid bilayers were examined using atomic force microscopy. A bilayer electrophysiological setup allowed for measurements of membrane conductance and capacitance. Here we demonstrate that the interaction of these proteins to form a fusion pore is dependent on the presence of t-SNAREs and v-SNARE in opposing bilayers. Addition of purified recombinant v-SNARE to a t-SNARE-reconstituted lipid membrane increased only the size of the globular t-SNARE oligomer without influencing the electrical properties of the membrane. However when t-SNARE vesicles were added to a v-SNARE membrane, SNAREs assembles in a ring pattern and a stepwise increase in capacitance, and increase in conductance were observed. Thus, t- and v-SNAREs are required to reside in opposing bilayers to allow appropriate t-/v-SNARE interactions leading to membrane fusion.

Pathways and intermediates in forced unfolding of spectrin repeats.

Spectrin repeats are triple-helical coiled-coil domains found in many proteins that are regularly subjected to mechanical stress. We used atomic force microscopy technique and steered molecular dynamics simulations to study the behavior of a wild-type spectrin repeat and two mutants. The experiments indicate that spectrin repeats can form stable unfolding intermediates when subjected to external forces. In the simulations the unfolding proceeded via a variety of pathways. Stable intermediates were associated to kinking of the central helix close to a proline residue. A mutant stabilizing the central helix showed no intermediates in experiments, in agreement with simulation. Spectrin repeats may thus function as elastic elements, extendable to intermediate states at various lengths.

Multi-bead-and-spring model to interpret protein detachment studied by AFM force spectroscopy.

This article deals with the detachment of molecules (fibrinogen) from a surface studied experimentally with an atomic force microscope. The detachment (or rupture) forces are measured as a function of the retraction velocity and exhibit a clear dependence on this parameter, even though the interaction between the molecules and the surface are nonspecific. To interpret these data, a mechanical multi-bead-and-spring model is developed. It consists of one to several parallel, "molecular" springs connected to an extra spring representing the cantilever that is moved at constant velocity. The free end of each molecular spring terminates with a particle that interacts with the surface through a Lennard-Jones potential. This Brownian dynamics model is used to analyze the experimental findings. In the framework of this model, it appears that the fibrinogen molecule must be ascribed a stiffness much smaller than that of the cantilever. In addition, several bonds between the molecule and the surface must be taken into account for the range of the molecule-surface interaction not to be unrealistically small. In future work, this model will be extended to more complex mechanisms such as the detachment of cells from a surface.