In the soft grip of nature.

Biological grippers can inspire the development of a new class of versatile soft grippers in agrorobotics and beyond.

Note: multiplexed multiple-tau auto- and cross-correlators on a single field programmable gate array.

We introduce a new multiple-tau hardware correlator design for computing fluorescence correlation functions (CFs) in real time. Use of hardware resources is minimized by scheduling the computation of different segments of the CFs on a single correlator block. Simultaneous calculation of two multiple-tau autocorrelation (ACFs) and two cross-correlation functions (CCFs) is implemented in LabVIEW on a National Instruments field programmable gate array (FPGA) card with a minimal sampling time of 400 ns. Raw data are stored with a time resolution of 50 ns. The design can be easily adapted to other FPGA cards and extended to more inputs.

Protein disorder prevails under crowded conditions.

Crowding caused by the high concentrations of macromolecules in the living cell changes chemical equilibria, thus promoting aggregation and folding reactions of proteins. The possible magnitude of this effect is particularly important with respect to the physiological structure of intrinsically disordered proteins (IDPs), which are devoid of well-defined three-dimensional structures in vitro. To probe this effect, we have studied the structural state of three IDPs, α-casein, MAP2c, and p21(Cip1), in the presence of the crowding agents Dextran and Ficoll 70 at concentrations up to 40%, and also the small-molecule osmolyte, trimethylamine N-oxide (TMAO), at concentrations up to 3.6 M. The structures of IDPs under highly diluted and crowded conditions were compared by a variety of techniques, fluorescence spectroscopy, acrylamide quenching, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, fluorescence correlation spectroscopy (FCS), and far-UV and near-UV circular dichroism (CD) spectroscopy, which allow us to visualize various levels of structural organization within these proteins. We observed that crowding causes limited structural changes, which seem to reflect the functional requirements of these IDPs. α-Casein, a protein of nutrient function in milk, changes least under crowded conditions. On the other hand, MAP2c and, to a lesser degree, p21(Cip1), which carry out their functions by partner binding and accompanying partially induced folding, show signs of local structuring and also some global compaction upon crowded conditions, in particular in the presence of TMAO. The observations are compatible with the possible preformation of binding-competent conformations in these proteins. The magnitude of these changes, however, is far from that of the cooperative folding transitions elicited by crowding in denatured globular proteins; i.e., these IDPs do remain in a state of rapidly interconverting structural ensemble. Altogether, our results underline that structural disorder is the physiological state of these proteins.

Brownian dynamics simulation of DNA unrolling from the nucleosome.

Nucleosomes organize chromatin in eukaryotic cells at the lowest scale by wrapping the DNA double helix around a histone octamer. The mechanism by which this structure can be opened, giving access to DNA-processing enzymes, is of fundamental biological importance. Here we describe a new coarse-grained model based on the toroidal geometry of the nucleosome which allows the simulation of nucleosome stretching experiments with a Brownian dynamics algorithm including hydrodynamics. We obtain force-extension curves and calculate energy barriers and kinetic rate constants of the unrolling transition from rupture forces.

Escherichia coli low-copy-number plasmid R1 centromere parC forms a U-shaped complex with its binding protein ParR.

The Escherichia coli low-copy-number plasmid R1 contains a segregation machinery composed of parC, ParR and parM. The R1 centromere-like site parC contains two separate sets of repeats. By atomic force microscopy (AFM) we show here that ParR molecules bind to each of the 5-fold repeated iterons separately with the intervening sequence unbound by ParR. The two ParR protein complexes on parC do not complex with each other. ParR binds with a stoichiometry of about one ParR dimer per each single iteron. The measured DNA fragment lengths agreed with B-form DNA and each of the two parC 5-fold interon DNA stretches adopts a linear path in its complex with ParR. However, the overall parC/ParR complex with both iteron repeats bound by ParR forms an overall U-shaped structure: the DNA folds back on itself nearly completely, including an angle of approximately 150 degrees . Analysing linear DNA fragments, we never observed dimerized ParR complexes on one parC DNA molecule (intramolecular) nor a dimerization between ParR complexes bound to two different parC DNA molecules (intermolecular). This bacterial segrosome is compared to other bacterial segregation complexes. We speculate that partition complexes might have a similar overall structural organization and, at least in part, common functional properties.

Protein-protein interactions determined by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is an emerging technique where the interaction between biomolecules is detected through their correlated motion. It offers the advantage of high (single-molecule) sensitivity; independence of molecular orientation or distance; and simultaneous measurement of molecular interactions, concentrations, and mobilities. Here we introduce the principle of the technique and review some recent examples from the literature where FCS has been used with autofluorescent proteins for measuring protein-protein interactions and mobilities in living cells.

DNA damaging capability of hematoporphyrin towards DNAs of various accessibilities.

In this work we wanted to verify that photoactivation of DNA-non-binding porphyrin derivative hematoporphyrin IX (Hp) is able to induce damages in DNAs of various accessibilities such as B-conformation and superhelical isolated DNA, nucleoprotein complex and intracellular DNAs. It was found that photodynamic reaction of Hp results significant changes in thermal stability of isolated T7 DNA and induces single strand breaks in supercoiled Bluescript plasmid isolated from Escherichia coli cells. As optical melting measurements revealed, the irradiation of photosensitized T7 nucleoprotein complex leads to a destabilization of the protein capsid. The photodynamic reaction affected both the protein structure and DNA-protein interaction, however, the parameters corresponding to the DNA denaturation are not influenced. The accumulation of Hp in HeLa cells was followed by laser scanning confocal microscopy. The picture received is typical for lipophilic dyes. When Hp loaded cells were irradiated, a reduction of viability could be observed in a concentration and a light dose dependent manner; 12microM porphyrin induced almost complete cell killing after 30min irradiation. After similar treatment, alkaline agarose gel electrophoresis of isolated nuclear DNA did not show the presence of single strand breaks. The alkaline comet assay also failed to demonstrate any DNA damage in HeLa cells. We also considered the possibility of the generation of damages in intracellular SV40 DNA. According to the electropherograms there was no difference between the patterns of DNAs from treated and control samples.

Polymer chain models of DNA and chromatin.

Many properties of the genome in the cell nucleus can be understood by modeling DNA and chromatin as a flexible polymer chain. This article introduces into current models for such a coarse-grained description and reviews some recent results from our own group. Examples given are the unrolling of DNA from the histone core and the response of the 30 nm chromatin fiber to mechanical stretching.

Abrogation of delayed type hypersensitivity response to Candida albicans produced by a molecular mimic of phosphorylated prolactin.

The effects of two major forms of prolactin (PRL) were examined on delayed type hypersensitivity (DTH) responses to Candida albicans. Unmodified PRL (U-PRL) had no effect on the DTH response, whereas a molecular mimic of phosphorylated PRL (S179D PRL) significantly inhibited immune responses to this robust antigen. This effect of S179D PRL was not accompanied by gross alterations in splenic T cell numbers, CD4/CD8 ratios, or T and B cell activation markers, but did produce a decrease in splenocyte apoptosis. Using gld animals, Fas ligand (FasL) was implicated in the suppressive effects of S179D PRL. Circulating IgG1 and IgG2 antibody levels were increased in response to treatment with both forms of PRL, but the effects of S179D PRL were most pronounced. Cytokine changes in the popliteal lymph nodes specific to S179D PRL treatment showed an inhibition of pro-inflammatory cytokines. In conclusion, mice treated with a molecular mimic of phosphorylated prolactin showed a profound inhibition of DTH responses to Candida correlating with an absence of GM-CSF, IL-4, and IL-13 production and a marked reduction in IL-12p70 synthesis.

Characterization of simian virus 40 on its infectious entry pathway in cells using fluorescence correlation spectroscopy.

SV40 (simian virus 40) is a double-stranded DNA virus and is strongly oncogenic in experimental animals. SV40 enters cells by atypical endocytosis mediated by caveolae, transporting the virus to its usual destination, namely the endoplasmic reticulum. The cellular mechanisms of capsid disassembly (uncoating) and deliverance of the viral genome into the cellular nucleus remain unknown. Here, we study (i) the formation of caveolae after viral infection and the diffusion of caveosome vesicles in the cytoplasm and (ii) the capsid disassembly and the mobility of the viral genome on its way to the nucleus, using fluorescence correlation spectroscopy. To follow the viral genome and capsids separately, the histone components of SV40 minichromosomes were labelled with enhanced yellow fluorescent protein and the capsid was labelled with a fluorescent red dye, Alexa568. We characterized the diffusion of caveosomes, the capsid disassembly process in the cytoplasm and the mobility of the viral genome in the nucleus, using two kinds of permissive cells.

Assessing the flexibility of intermediate filaments by atomic force microscopy.

Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a approximately 45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (approximately 3-10 microm) and microtubules (approximately 1-8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing. Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 microm and 1 microm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be approximately 1 microm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support. Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.

Mad dogs, Englishmen and apoptosis: the role of cell death in UV-induced skin cancer.

Apoptosis plays a critical role in the development and progression of ultraviolet-induced skin cancers. In particular, Fas and Fas ligand (FasL) interactions are known to control the development of "sunburn cells" or apoptotic keratinocytes in the UV-exposed epidermis. In the absence of functional Fas/FasL signaling, UV-induced apoptosis is diminished and mutations rapidly accumulate. UV-induced suppression of host immunity, a process regulating skin cancer outgrowth, is also controlled through Fas/FasL interactions. Other death receptors, such as the receptor for tumor necrosis factor, may also contribute to UV-induced carcinogenesis and progression. Understanding the involvement of cell death in cancers caused by exposure to sunlight may provide novel approaches for prevention and therapy of these ever-increasing malignancies.

Detection of NGF-receptors TrkA and p75NTR in human tumor cell lines and effect of NGF on the growth characteristic of the UT-7/EPO cell line.

The receptors for nerve growth factor (NGF)--TrkA and p75NTR--were detected at the mRNA and the protein level in various human tumor cell lines. The NGF receptor TrkA was found on all examined tumor cell lines and is not restricted to cells belonging to the nervous system. NGF did not influence the proliferation rate of TrkA-positive cells NMB, K562, UT-7/EPO and PC-12. After NGF induction, the production level of the differentiation marker c-fos was increased in UT-7/EPO and PC- 12 cells. NGF-treatment of the UT-7/EPO cells and deprivation of erythropoietin (EPO) led to the new adherent cell line UT-7/NGF. Although UT-7/NGF showed a similar growth curve as UT-7/EPO, there were differences in the pattern of adhesion molecules and of the cytoskeleton. The effect of NGF on the cytoskeleton could not be induced in other human cell lines like NMB or KTCTL-30. TrkA inhibition with K252a--a blocker of Trk-induced receptor kinase--suggests, that the NGF signal may be transduced by the TrkA receptor in UT-7/NGF cells. This indicates that NGF is a decisive mediator of cellular adhesion.

Intrachain reactions of supercoiled DNA simulated by Brownian dynamics.

We considered an irreversible biochemical intrachain reaction of supercoiled DNA as a random event that occurs, with certain probability, at the instant of collision between two reactive groups bound to distant DNA sites. Using the Brownian dynamics technique, we modeled this process for a supercoiled DNA molecule of 2.5 kb length in dilute aqueous solution at an NaCl concentration of 0.1 M. We calculated the mean reaction time tau(Sigma) as a function of the intrinsic second-order rate constant k(I), the reaction radius R, and the contour separation S of the reactive groups. At the diffusion-controlled limit (k(I) --> infinity), the kinetics of reaction are determined by the mean time tau(F) of the first collision. The dependence of tau(F) on R is close to inversely proportional, implying that the main contribution to the productive collisions is made by bending of the superhelix axis. At sufficiently small k(I), the mean reaction time can be satisfactory approximated by tau(Sigma) = tau(F)(app) + 1/(k(I)c(L)), where c(L) is the local concentration of one reactive group around the other, and tau is an adjustable parameter, which we called the apparent time of the first collision. The value of tau depends on R very weakly and is approximately equal to the mean time of the first collision caused by mutual reptation of two DNA strands forming the superhelix. The quasi-one-dimensional reptation process provides the majority of productive collisions at small k(I) values.

Trajectory of nucleosomal linker DNA studied by fluorescence resonance energy transfer.

While the structure of the nucleosome core is known in atomic detail, the precise geometry of the DNA beyond the core particle is still unknown. We have used fluorescence resonance energy transfer (FRET) for determining the end-to-end distance of DNA fragments assembled with histones into nucleosomes. The DNA of a length of 150-220 bp was labeled with rhodamine-X on one end and fluorescein or Alexa 488 on the other. Assembling nucleosomes on these DNA fragments leads to a measurable energy transfer. The end-to-end distance computed from the FRET increases from 60 +/- 5 A at 150 bp to 75 +/- 5 A at 170 bp without measurable change above it. These distances are compatible with different geometries of the linker DNA, all having in common that no crossing can be observed up to 220 bp. Addition of H1 histone leads to an increase in energy transfer, indicating a compaction of the linker DNA toward the nucleosome.

Rotational dynamics of curved DNA fragments studied by fluorescence polarization anisotropy.

The rotational dynamics of short DNA fragments with or without intrinsic curvature were studied using time-resolved phase fluorimetry of intercalated ethidium with detection of the anisotropy. Parameters determined were the spinning diffusion coefficient of the DNA fragments about the long axis and the zero-time ethidium fluorescence anisotropy. We find a significant decrease in the spinning diffusion coefficient for all curved fragments compared to the straight controls. This decrease is likewise evident in rotational diffusion coefficients computed from DNA structures obtained by a curvature prediction program for these sequences. Using a hinged-cylinder model, we can identify the change in rotational diffusion coefficient with a permanent bend of 13-16 degrees per helix turn for the sequences studied. Moreover, for some of the curved fragments an increased flexibility has to be assumed in addition to the permanent bend in order to explain the data.

Diffusion-controlled intrachain reactions of supercoiled DNA: Brownian Dynamics simulations.

The Brownian Dynamics technique was used to model a diffusion-controlled intramolecular reaction of supercoiled DNA (2500 basepairs) in 0.1 M sodium chloride solution. The distance between the reactive groups along the DNA contour was 470 basepairs. The reaction radius was varied from 6 to 20 nm. The results are presented in terms of the probability distribution P(F)(t) of the first collision time. The general form of the function P(F)(t) could be correctly predicted by a simple analytical model of one-dimensional diffusion of the superhelix ends along the DNA contour. The distribution P(F)(t) is essentially non-exponential: within a large initial time interval, it scales as P(F)(t) approximately t(-1/2), which is typical for one-dimensional diffusion. However, the mean time of the first collision is inversely proportional to the reaction radius, as in three dimensions. A visual inspection of the simulated conformations showed that a considerable part of the collisions is caused by the bending of the superhelix axis in the regions of the end loops, where the axis is most flexible. This fact explains why the distribution P(F)(t) combines the features of one- and three-dimensional diffusion. The simulations were repeated for a DNA chain with a permanent bend of 100 degrees in the middle position between the reactive groups along the DNA contour. The permanent bend changes dramatically the form of the distribution P(F)(t) and reduces the mean time of the first collision by approximately one order of magnitude.

Computation of writhe in modeling of supercoiled DNA.

We describe four previously unpublished methods allowing the computation of the writhe for a supercoiled DNA molecule modeled by a polymer chain consisting of straight segments. These methods are compared with each other in terms of computational efficiency and the scope of their applicability is discussed.

The effect of the DNA conformation on the rate of NtrC activated transcription of Escherichia coli RNA polymerase.sigma(54) holoenzyme.

The transcription activator protein NtrC (nitrogen regulatory protein C) can catalyze the transition of Escherichia coli RNA polymerase complexed with the sigma 54 factor (RNAP.sigma(54)) from the closed complex (RNAP.sigma(54) bound at the promoter) to the open complex (melting of the promoter DNA). This process involves phosphorylation of NtrC (NtrC-P), assembly of an octameric NtrC-P complex at the enhancer sequence, interaction of this complex with promoter-bound RNAP.sigma(54) via DNA looping, and hydrolysis of ATP. We have used this system to study the influence of the DNA conformation on the transcription activation rate in single-round transcription experiments with superhelical plasmids as well as linearized templates. Most of the templates had an intrinsically curved DNA sequence between the enhancer and the promoter and differed with respect to the location of the curvature and the distance between the two DNA sites. The following results were obtained: (i) a ten- to 60-fold higher activation rate was observed with the superhelical templates as compared to the linearized conformation; (ii) the presence of an intrinsically curved DNA sequence increased the activation rate of linear templates about five times; (iii) no systematic effect for the presence and/or location of the inserted curved sequence was observed for the superhelical templates. However, the transcription activation rate varied up to a factor of 10 between some of the constructs. (iv) Differences in the distance between enhancer and promoter had little effect for the superhelical templates studied. The results were compared with theoretical calculations for the dependence of the contact probability between enhancer and promoter expressed as the molar local concentration j(M). A correlation of j(M) with the transcription activation rate was observed for values of 10(-8) M

Physical characterization of plakophilin 1 reconstituted with and without zinc.

Plakophilin 1 (PKP1) belongs to the arm-repeat protein family which is characterized by the presence of a conserved 42-amino-acid motif. Despite individual members of the family containing a similar type of structural domain, they exhibit diverse cellular functions. PKP1 is ubiquitously expressed in human tissues and, depending on the type of cell, found prominently in the karyoplasm and/or in desmosomes. In surface plasmon resonance detection experiments, we noticed that PKP1 specifically bound zinc but not calcium or magnesium. Therefore we have used circular dichroism spectroscopy, limited proteolysis, analytical ultracentrifugation, electron microscopy and dynamic light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc. The alpha helix content of PKP1 was considerably higher when reconstituted with zinc than without. By atomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly associated with one molecule of wild-type PKP1. The zinc-reconstituted protein formed globular particles of 21.9 +/- 8.4 nm diameter, as measured by electron microscopy after glycerol spraying/rotary metal shadowing. In parallel, the average sedimentation coefficient (s20, w) for zinc-containing PKP1 was 41S and its diffusion coefficient, as obtained by dynamic light scattering, 1.48 x 10-7 cm2.s-1. The molecular mass of 2.44 x 106 obtained from s and D yields an average stoichiometry of 30 for the PKP1 oligomer. In contrast, PKP1, reconstituted without zinc, contained no significant amount of zinc, sedimented with 4.6S, and was present in monomeric form as determined by sedimentation equilibrium centrifugation.