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°. For DNA with bulged adenosines, we also quantified the kink angles introduced by the insertion of 1, 3 and 5 bases to be 32°؎ 6°, 56°؎ 4°a nd 73 ؎ 2°, respectively. Moreover, the rotation angles and shifts of the helices were calculated to describe the relative orientation of the two arms in detail.absolute distance measurements ͉ fluorescence energy transfer ͉ multiparameter fluorescence detection ͉ nucleic acid structures I n recent years, fluorescence energy transfer experiments (FRET) have shown great potential for subnanometer analysis of biomolecular structures and their dynamics that can even be applied to single molecules (1-5). Calculation of absolute FRET distances between a donor and acceptor fluorophore is complicated because of several ''calibration'' factors such as detection efficiencies, spectral cross-talk and fluorescence quantum yields (6) that are difficult to determine accurately. Multiparameter Fluorescence Detection (MFD) (7) avoids most pitfalls (5) by simultaneously collecting all fluorescence parameters (intensity, lifetime and anisotropy in both spectral ranges) at the singlemolecule level.However, determination of absolute distances remains a major challenge because of the uncertainty in the fluorophore positions. This uncertainty is due to the use of the long linkers through which the fluorophores are attached to the biomolecules. Orientational freedom is a prerequisite to safely assume an orientation factor ( 2 ) of 2/3 (8); however, this prevents a defined fluorophore position, which is needed for fitting data to a geometric model. Here, we present a new conversion function for experimental FRET efficiencies that considers the dynamics of the movement of the fluorop...

Influenza virus belongs to a wide range of enveloped viruses. The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the millimolar range [Sauter NK, et al. (1992) Biochemistry 31:9609-9621], indicating a multivalent binding mode. Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy-based single-molecule force spectroscopy revealed very low interaction forces. Further, the observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor, allowing rationalization of influenza virus-cell binding quantitatively at the molecular level.multivalency | adhesion | avidity | tropism

Metallic nanoparticles, in particular gold nanoparticles (AuNPs), offer a wide spectrum of applications in biomedicine. A crucial issue is their cytotoxicity, which depends greatly on various factors, including morphology of nanoparticles. Because metallic nanoparticles have an effect on cell membrane integrity, their shape and size may affect the viability of cells, due to their different geometries as well as physical and chemical interactions with cell membranes. Variations in the size and shape of gold nanoparticles may indicate particular nanoparticle morphologies that provide strong cytotoxicity effects. Synthesis of different sized and shaped bare AuNPs was performed with spherical (~ 10 nm), nanoflowers (~ 370 nm), nanorods (~ 41 nm), nanoprisms (~ 160 nm) and nanostars (~ 240 nm) morphologies. These nanostructures were characterized and interacting with cancer (HeLa) and normal (HEK293T) cell lines and cell viability tests were performed by WST-1 tests and fluorescent live/dead cell imaging experiments. It was shown that various shapes and sizes of gold nanostructures may affect the viability of the cells. Gold nanospheres and nanorods proved to be more toxic than star, flower and prism gold nanostructures. This may be attributed to their small size and aggregation process. This is the first report concerning a comparison of cytotoxic profile in vitro with a wide spectrum of bare AuNPs morphology. The findings show their possible use in biomedical applications.

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. Its C-terminal region, the receptor-binding domain (RD), contains ∼40 calcium-binding Repeat in ToXin (RTX) motifs, which are characteristic of many virulence factors of pathogenic bacteria. We previously showed that RD is intrinsically disordered in the absence of calcium and acquires its functional three-dimensional structure upon calcium binding. To gain further insight into the physicochemical properties of RD, we characterized its calcium-induced conformational and stability changes by combining spectroscopic approaches. We show that RD, in the absence of calcium, adopts premolten globule conformations, due in part to the strong internal electrostatic repulsions between the negative charges of the aspartate-rich polypeptide sequence. Accordingly, sodium is able to screen these electrostatic repulsions, allowing a partial compaction of the polypeptide, whereas calcium triggers a strong compaction as well as the acquisition of secondary and tertiary structures in a highly cooperative manner. The differential sensitivity of the calcium-loaded state to guanidinium- and urea-induced denaturations provides further evidence that electrostatic interactions play a critical role in the folding and stability of RD. These results provide new insights into the folding/function relationship of the RTX motifs.