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Biomedical subjects

A Libchaber

Publications and source records attributed to A Libchaber.

At least 19 recordsLinked to original sources
PubMed1999

RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis.

The polymerization of RecA on individual double-stranded DNA molecules is studied. A linear DNA (lambda DNA, 48.5 Kb), anchored at one end to a cover glass and at the other end to an optically trapped 3-micrometers diameter polystyrene bead, serves as a template. The elongation caused by RecA assembly is measured in the presence of ATP and ATP[gammaS]. By using force extension and hydrodynamic recoil, a value of the persistence length of the RecA-DNA complex is obtained. In the presence of ATP, the polymer length is unstable, first growing to saturation and then decreasing. This suggests a transient dynamics of association and dissociation for RecA on a double-stranded DNA, the process being controlled by ATP hydrolysis. Part of this dynamics is suppressed in the presence of ATP[gammaS], leading to a stabilized RecA-DNA complex. A one-dimensional nucleation and growth model is presented that may account for the protein assembly.

Adenosine Triphosphate↗
PubMed1999

Thermodynamic basis of the enhanced specificity of structured DNA probes.

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

Base Sequence↗
PubMed1998

Kinetics of conformational fluctuations in DNA hairpin-loops.

The kinetics of DNA hairpin-loop fluctuations has been investigated by using a combination of fluorescence energy transfer and fluorescence correlation spectroscopy. We measure the chemical rates and the activation energies associated with the opening and the closing of the hairpin for different sizes and sequences of the loop and for various salt concentrations. The rate of unzipping of the hairpin stem is essentially independent of the characteristics of the loop, whereas the rate of closing varies greatly with the loop length and sequence. The closing rate scales with the loop length, with an exponent 2.6 +/- 0.3. The closing rate is increased at higher salt concentrations. For hairpin closing, a loop of adenosine repeats leads to smaller rates and higher activation energies than a loop with thymine repeats.

DNA↗
PubMed1998

Gramicidin channel kinetics under tension.

We have measured the effect of tension on dimerization kinetics of the channel-forming peptide gramicidin A. By aspirating large unilamellar vesicles into a micropipette electrode, we are able to simultaneously monitor membrane tension and electrical activity. We find that the dimer formation rate increases by a factor of 5 as tension ranges from 0 to 4 dyn/cm. The dimer lifetime also increases with tension. This behavior is well described by a phenomenological model of membrane elasticity in which tension modulates the mismatch in thickness between the gramicidin dimer and membrane.

Dimerization↗
PubMed1997

DNA solution of the maximal clique problem.

The maximal clique problem has been solved by means of molecular biology techniques. A pool of DNA molecules corresponding to the total ensemble of six-vertex cliques was built, followed by a series of selection processes. The algorithm is highly parallel and has satisfactory fidelity. This work represents further evidence for the ability of DNA computing to solve NP-complete search problems.

Algorithms↗
PubMed1997

Parallel overlap assembly for the construction of computational DNA libraries.

Algorithms for computing with DNA currently require the construction of pools of molecules in which each distinct molecule represents a different starting point for the calculation. We have begun building such pools using the technique of parallel overlap assembly that is already used for the generation of diversity in biologically useful combinatorial search techniques such as gene shuffling. Unlike these applications, a pool in a molecular computer must be complete, containing all possible strands, and ordered, having minimal contamination from incorrectly assembled DNA. We present an experiment in which parallel overlap assembly is used to construct a computational pool and an experiment in which this pool is used to solve the NP-complete maximal-clique problem.

Algorithms↗
PubMed1997

Elasticity and structure of eukaryote chromosomes studied by micromanipulation and micropipette aspiration.

The structure of mitotic chromosomes in cultured newt lung cells was investigated by a quantitative study of their deformability, using micropipettes. Metaphase chromosomes are highly extensible objects that return to their native shape after being stretched up to 10 times their normal length. Larger deformations of 10 to 100 times irreversibly and progressively transform the chromosomes into a "thin filament," parts of which display a helical organization. Chromosomes break for elongations of the order of 100 times, at which time the applied force is around 100 nanonewtons. We have also observed that as mitosis proceeds from nuclear envelope breakdown to metaphase, the native chromosomes progressively become more flexible. (The elastic Young modulus drops from 5,000 +/- 1,000 to 1,000 +/- 200 Pa.) These observations and measurements are in agreement with a helix-hierarchy model of chromosome structure. Knowing the Young modulus allows us to estimate that the force exerted by the spindle on a newt chromosome at anaphase is roughly one nanonewton.

Anaphase↗
PubMed1995

Flexibility of myosin attachment to surfaces influences F-actin motion.

We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin-coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4-5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement.

Actins↗
PubMed1995

Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.

We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement.

Actins↗