Biomedical subjects

T A Duke

Publications and source records attributed to T A Duke.

6 recordsLinked to original sources

PubMed2001

Separation of 100-kilobase DNA molecules in 10 seconds.

Long double-stranded DNA molecules were separated in microfabricated hexagonal arrays in less than 1 min, several orders of magnitude faster than by using conventional technology. DNA samples were first concentrated at the entrance to the array in a thin band by entropic focusing. They were then separated by pulsed field electrophoresis. T4 (168.9 kbp) and lambda (48.5 kbp) DNAs could be resolved into two clearly separated bands in approximately 10 s in these experiments. This corresponds to a mass resolution of 6% in 11 min in a 1-cm-long array.

Algorithms↗

PubMed2001

Conformational spread in a ring of proteins: a stochastic approach to allostery.

We recently suggested that the sensitivity and range of a cluster of membrane receptors in bacteria would be enhanced by cooperative interactions between neighbouring proteins. Here, we examine the consequences of this "conformational spread" mechanism for an idealised one-dimensional system comprising a closed ring of identical allosteric protomers (protein molecules, or a group of protein domains operating as a unit). We show analytically and by means of Monte Carlo simulations that a ring of allosteric protomers can exhibit a switch-like response to changes in ligand concentration. We derive expressions for the sensitivity and cooperativity of switching and show that the maximum sensitivity is proportional to the number of protomers in the ring. A ring of this kind can reproduce the sensitivity and kinetics of the switch complex of a bacterial flagellar motor, for example, which is based on a ring of 34 FliM proteins. We also compare smaller rings of conformationally coupled protomers to classical allosteric proteins such as haemoglobin and show that the canonical MWC and KNF models arise naturally as limiting cases. Conformational spread appears to be a natural extension of the familiar mechanism of allostery: a physically realistic mechanism that should apply widely to many structures built from protein molecules.

Allosteric Regulation↗

PubMed1999

Sorting by diffusion: an asymmetric obstacle course for continuous molecular separation.

A separation technique employing a microfabricated sieve has been demonstrated by observing the motion of DNA molecules of different size. The sieve consists of a two-dimensional lattice of obstacles whose asymmetric disposition rectifies the Brownian motion of molecules driven through the device, causing them to follow paths that depend on their diffusion coefficient. A nominal 6% resolution by length of DNA molecules in the size range 15-30 kbp may be achieved in a 4-inch (10-cm) silicon wafer. The advantage of this method is that samples can be loaded and sorted continuously, in contrast to the batch mode commonly used in gel electrophoresis.

Chemical Fractionation↗

PubMed1999

Heightened sensitivity of a lattice of membrane receptors.

Receptor proteins in both eukaryotic and prokaryotic cells have been found to form two-dimensional clusters in the plasma membrane. In this study, we examine the proposition that such clusters might show coordinated responses because of the spread of conformational states from one receptor to its neighbors. A Monte Carlo simulation was developed in which receptors flipped in probabilistic fashion between an active and an inactive state. Conformational energies depended on (i) ligand binding, (ii) a chemical modification of the receptor conferring adaptation, and (iii) the activity of neighboring receptors. Rate constants were based on data from known biological receptors, especially the bacterial Tar receptor, and on theoretical constraints derived from an analogous Ising model. The simulated system showed a greatly enhanced sensitivity to external signals compared with a corresponding set of uncoupled receptors and was operational over a much wider range of ambient concentrations. These and other properties should make a lattice of conformationally coupled receptors ideally suited to act as a "nose" by which a cell can detect and respond to extracellular stimuli.

Binding Sites↗

PubMed1999

Molecular model of muscle contraction.

A quantitative stochastic model of the mechanochemical cycle of myosin, the protein that drives muscle contraction, is proposed. It is based on three premises: (i) the myosin head incorporates a lever arm, whose equilibrium position adjusts as each of the products of ATP hydrolysis dissociates from the nucleotide pocket; (ii) the chemical reaction rates are modified according to the work done in moving the arm; and (iii) the compliance of myosin's elastic element is designed to permit many molecules to work together efficiently. The model has a minimal number of parameters and provides an explanation, at the molecular level, of many of the mechanical and thermodynamic properties of steadily shortening muscle. In particular, the inflexion in the force-velocity curve at a force approaching the isometric load is reproduced. Moreover, the model indicates that when large numbers of myosin molecules act collectively, their chemical cycles can be synchronized, and that this leads to stepwise motion of the thin filament. The oscillatory transient response of muscle to abrupt changes of load is interpreted in this light.

Animals↗

PubMed1996

Pulsed-field electrophoresis in microlithographic arrays.

Transverse pulsed-field electrophoresis of DNA has been conducted in a silicon array engineered by optical lithography and the motion of individual molecules observed by fluorescence microscopy. In strong fields, the molecules can be maintained in highly stretched, linear conformations. When the field is switched through an obtuse angle, they head off in the new direction led by what was formerly their tail end. This backtracking gives rise to fractionation that is linear with molecular weight. A simple prescription exists for choosing the field parameters to obtain a particular range of separation. Since the molecular motions are much more uniform than those that occur in a gel, it is anticipated that the arrays will permit more efficient fractionation than traditional pulsed-field gel electrophoresis. Arrays suitably scaled down in size may be useful for pulsed-field sequencing.

DNA↗