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Biomedical subjects

R H Austin

Publications and source records attributed to R H Austin.

53 records · Page 3Linked to original sources
PubMed1983

Rotational diffusion of Escherichia coli RNA polymerase free and bound to deoxyribonucleic acid in nonspecific complexes.

We have studied the rotational diffusion of Escherichia coli RNA polymerase free in solution and bound nonspecifically to DNA fragments. The rotational motion was measured by the decay in anisotropy of the triplet-triplet absorption by using as probes either the liganded enzyme inhibitor Rose Bengal or eosin 5'-isothiocyanate conjugated to the protein. The time resolution extended from 10 ns to 1 ms. Free RNA polymerase (holoenzyme) at high salt concentration (1 M NaCl) is monomeric and diffuses at 5 degrees C with a rotational correlation time of 0.66 microseconds, corresponding to an equivalent hydrodynamic sphere with a radius of 7.4 nm. These values and the known molecular weight are most compatible with a nonspherical shape, e.g., an oblate ellipsoid with an axial ratio of about 3. In 0.1 M NaCl, the holoenzyme is dimeric and has a rotational correlation time of 2 microseconds. The decay of anisotropy is at least biexponential upon binding RNA polymerase to calf thymus DNA or to poly[d(A-T)]. The fast component with half of the amplitude has decay kinetics comparable to those seen with the free monomeric enzyme. The slow component has a rotational correlation time of about 14 microseconds and is independent of DNA chain length in the range greater than 180 base pairs. Both rotational correlation times decrease with temperature, and the relative amplitudes change such that the faster component dominates at higher temperature. The rotational relaxation of the enzyme-DNA complexes is discussed in terms of alternative models involving rigid rod-sphere diffusion, conformational changes in the enzyme and/or DNA, sliding motions of the protein along the DNA, and torsional-bending motions of DNA envisioned as a deformable rod.

Circular Dichroism↗
PubMed1983

Triplet anisotropy decay measurements of DNA internal motion.

Triplet anisotropy decay techniques have been used to measure the internal flexibility and overall rotational motions of DNA over a time range of 15 ns to 200 microseconds. Nearly monodisperse DNA fragments with lengths varying from 65 to 600 base pairs were studied using the intercalating dye methylene blue as a triplet probe. The slow end-over-end tumbling of short DNA fragments (less than 165 base pairs) is as predicted for a rigid rod. As expected, a longer DNA fragment (600 base pairs) experiences slow segmental motions of its helix axis. At the earliest times, anisotropy decays more rapidly than expected for a rigid rod, suggesting that, when it is bound, methylene blue monitors fast internal motions of the helix. Since the rod-like end-over-end tumbling rules out fast bending motions (for short DNA fragments), the fast components of DNA anisotropy decay must be due to twisting motions of the helix, occurring with a time constant near 50 ns. The same techniques were used to measure the conformational flexibility of DNA in the nucleosome. It is concluded that, when the DNA helix is wrapped to form a nucleosome, it experiences substantial internal flexibility, occurring with a time constant near 30 ns. The amplitude and time-scale of this motion appear to be similar to that seen in the uncomplexed DNA helix.

Animals↗
PubMed1982

Molecular motion of DNA as measured by triplet anisotropy decay.

We have used triplet anisotropy decay techniques to measure the internal flexibility and overall rotational motion of DNA, covering a time range from 15 ns to 200 mus. Nearly monodisperse DNA fragments 65--600 base pairs long were studied by using the intercalating dye methylene blue as a triplet probe. We found that the slow end-over-end tumbling of short DNA fragments (less than or equal to 165 base pairs) is as predicted for a rigid rod. As expected, a longer DNA fragment (600 base pairs) experiences slow segmental motion of its helix axis. We found that, at the earliest times, anisotropy decays more rapidly than expected for a rigid rod, suggesting that, when bound, methylene blue monitors fast internal motion of the helix. Since the rod-like end-over-end tumbling of short fragments rules out fast bending motions, we conclude that the fast components of DNA anisotropy decay are due to twisting motion of the helix, occurring with a time constant near 50 ns.

DNA↗
PubMed1982

DNA motions in the nucleosome core particle.

We have used time-resolved triplet state anisotropy decay techniques to measure the conformational flexibility of DNA in the nucleosome. From these measurements we conclude that, in a nucleosome, the DNA helix experiences substantial internal flexibility, which occurs with a time constant near 30 nsec. We find that our data can be fit well by a modified version of the Barkley-Zimm model for DNA motion, allowing only DNA twisting motions and the overall tumbling of the nucleosome. That fit yields a calculated torsional rigidity equal to 1.8 X 10(-19) erg X cm, a value equal to that measured for uncomplexed DNA. We conclude from such similarity that large, fast twisting motions of the DNA helix persist, nearly unaltered, when DNA is wrapped to form a nucleosome.

Animals↗
PubMed1981

Hemerythrin's oxygen-binding reaction studied by laser photolysis.

The dioxygen--iron bond in oxyhemerythrin is shown to be photosensitive. The recombination reaction after photodissociation depends strongly on solvent viscosity. In water (eta = 1 centipoise or 1 x 10(-3) Pa . s) the recombination is monophasic and second-order in solvent oxygen concentration, with a bimolecular rate coefficient of 2.9 x 10(7) M-1 s-1. In a glycerol/water mixture (eta = 180 centipoise) a concentration-dependent geminate recombination process is also seen. This opens a class of proteins to study by flash photolysis.

Animals↗
PubMed1979

The rotational diffusion of cytochrome b5 in lipid bilayer membranes. Influence of the lipid physical state.

A derivative of the integral membranes protein, cytochrome b5, has been prepared in which the native heme group has been replaced by the structurally similar rhodium(III)-protoporphyrin IX. This metalloporphyrin has a finite triplet yield with a single exponential decay time of 22 microsecond in water. After insertion of the metalloporphyrin into the protein, its triplet-state decay becomes strongly nonexponential with at least three equal amplitude components with time constants varying over a range of 100. The derivatized protein has been incorporated into unilamellar liposomes prepared from dimyristoyllecithin, and the rotational diffusion of the protein in the lipid bilayer has been studied at temperatures above and below the lipid phase transition temperature via triplet absorbance anisotropy decay. The anisotropy decay curves are biphasic both above and below the lipid phase transition. The rotational diffusion constant is found to be 2.4 X 10(5) s-1 at 35 degrees C, and 1.1 X 10(4) s-1 at 10 degrees C, both being calculated from the fast decay component. The ratio of the limiting anisotropy to the initial anisotropy is 0.6 at both temperatures. This implies a cone of restricted motion of 34 degrees for the protein in the bilayer.

Animals↗
PubMed1979

Rotational diffusion of cell surface components by time-resolved phosphorescence anisotropy.

The rotational diffusion of concanavalin A receptors of viable Friend erythroleukemia cells and the band 3 anion transport system of human erythrocytes has been measured via the time-dependent phosphorescence emission intensity and anisotropy of triplet probes excited by a 5-ns laser pulse. High-quality phosphorescence decay curves with a 10-microseconds time resolution were obtained at concentrations of the eosin probe down to 20 nM and in aqueous media at temperatures of 4-38 degrees C. A strong temperature dependence in the rotational behavior was observed for the band 3 anion transport protein, but the lectin receptors of the Friend erythroleukemia cells were found to be immobile on the time scale of 1-4000 microseconds at either 4 degrees C or 37 degrees C. The technique is applicable to other triplet probes and membrane components of living cells under conditions that do not destroy viability.

Concanavalin A↗
PubMed1978

The rate of entry of dioxygen and carbon monoxide into myoglobin.

The model for carbon monoxide or dioxygen recombination with heme proteins developed by the group at the University of Illinois is reexamined. We propose that the carbon monoxide or dioxygen molecule enters the protein at essentially a diffusion-limited rate determined by the solvent viscosity and that the protein offers no important barriers to this entry. The viscosity dependence of the entry rate k(ED), its magnitude (1 x 10(10) M(-1)s(-1), and the rate of quenching of triplet states of protoprophyrin IX in apomyoglobin by dioxygen are used as supporting evidence. Comparison is made to the model of a fluctuating protein developed by G. Weber.

Carbon Monoxide↗
PubMed1978

Fast reactions in carbon monoxide binding to heme proteins.

Using fast flash photolysis, we have measured the binding of CO to carboxymethylated cytochrome c and to heme c octapeptide as a function of temperature (5 degrees-350 degreesK) over an extended time range (100 ns(-1) ks). Experiments used a microsecond dye laser (lambda = 540 nm), and a mode-locked frequency-doubled Nd-glass laser (lambda = 530 nm). At low temperatures (5 degrees-120 degreesK) the rebinding exhibits two components. The slower component (I) is nonexponential in time and has an optical spectrum corresponding to rebiding from an S = 2, CO-free deoxy state. The fast component (I*) is exponential in time with a lifetime shorter than 10 mus and an optical spectrum different from the slow component. In myoglobin and the separated alpha and beta chains of hemoglobin, only process I is visible. The optical absorption spectrum of I* and its time dependence suggest that it may correspond to recombination from an excited state in which the iron has not yet moved out of the heme plane. The temperature dependences of both processes have been measured. Both occur via quantum mechanical tunneling at the lowest temperatures and via over-the-barrier motion at higher temperatures.

Binding Sites↗
PubMed1976

Tunneling in ligand binding to heme proteins.

Rebinding of carbon monoxide to the beta chain of hemoglobin after photodissociation by a laser flash is intramolecular below about 200 K. Above 25 K, rebinding occurs via classical over-the-barrier motion; below, quantum-mechanical tunneling dominates. Both are described by an energy spectrum peaked at Epeak=4.0 kilojoules per mole. The barrier width d(E), determined from the energy dependence of the tunneling rate, depends on barrier height, d(E) approximately 0.05 nanometer X (E/Epeak) 1.5.

Carbon Monoxide↗
PubMed1975

Dynamics of ligand binding to myoglobin.

Myoglobin rebinding of carbon monoxide and dioxygen after photodissociation has been observed in the temperature range between 40 and 350 K. A system was constructed that records the change in optical absorption at 436 nm smoothly and without break between 2 musec and 1 ksec. Four different rebinding processes have been found. Between 40 and 160 K, a single process is observed. It is not exponential in time, but approximately given by N(t) = (1 + t/to)-n, where to and n are temperature-dependent, ligand-concentration independent, parameters. At about 170 K, a second and at 200 K, a third concentration-independent process emerge. At 210 K, a concentration-dependent process sets in. If myoglobin is embedded in a solid, only the first three can be seen, and they are all nonexponential. In a liquid glycerol-water solvent, rebinding is exponential. To interpret the data, a model is proposed in which the ligand molecule, on its way from the solvent to the binding site at the ferrous heme iron, encounters four barriers in succession. The barriers are tentatively identified with known features of myoglobin. By computer-solving the differential equation for the motion of a ligand molecule over four barriers, the rates for all important steps are obtained. The temperature dependences of the rates yield enthalpy, entropy, and free-energy changes at all barriers. The free-energy barriers at 310 K indicate how myoglobin achieves specificity and order. For carbon monoxide, the heights of these barriers increase toward the inside; carbon monoxide consequently is partially rejected at each of the four barriers. Dioxygen, in contrast, sees barriers of about equal height and moves smoothly toward the binding site. The entropy increases over the first two barriers, indicating a rupturing of bonds or displacement of residues, and then smoothly decreases, reaching a minimum at the binding site. The magnitude of the decrease over the innermost barrier implies participation of heme and/or protein. The nonexponential rebinding observed at low temperatures and in solid samples implies that the innermost barrier has a spectrum of activation energies. The shape of the spectrum has been determined; its existence can be explained by assuming the presence of many conformational states for myoglobin. In a liquid at temperatures above about 230 K, relaxation among conformational states occurs and rebinding becomes exponential.

Binding Sites↗
PubMed1973

Dynamics of carbon monoxide binding by heme proteins.

Rebinding of carbon monoxide to myoglobin and to cytochrome P-450 after removal by a light flash occurs down to 50 degrees K for myoglobin and 25 degrees K for cytochrome P-450 in glycerol-water solution. Above 240 degrees K the reaction is second order; between 240 degrees and 200 degrees K the rebinding becomes exponential and independent of the carbon monoxide concentration. Below 150 degrees K the reaction follows a power law and is approximately 10(3) times faster for cytochrome P-450 than for myoglobin.

Carbon Monoxide↗
PubMed1972

Photoreduction of NADP + sensitized by synthetic pigment systems.

Two synthetic pigment systems capable of enzymatically photoreducing NADP(+) are described. One system contains proflavine; the other, acridine. The complete system consists of ethylene diamine tetraacetic acid (EDTA), proflavine (or acridine), ferredoxin-NADP reductase (EC 1.6.99.4), and NADP(+). The two pigments initiate the photoreduction of NADP(+) in different portions of the electromagnetic spectrum. Proflavine photosensitizes in the visible portion; acridine, in the ultraviolet. Neither proflavine nor acridine is structurally related to chlorophyll. The acridine system has the attractive property that the enzyme, ferredoxin-NADP reductase, is the only component of the system that absorbs appreciably in the visible region of the spectrum.

Acridines↗
PubMedDate not supplied

Evidence for kinks in DNA folding in the nucleosome.

The nucleosome subunit of chromatin consists of DNA folded around a histone core as a 1.8-turn left-handed solenoid. The crystal structure of the nucleosome core particle revealed that it has a dyad symmetry axis and that the minor helix groove faces outwards from the protein core. Richmond et al. noticed that the path traversed by the helix has severe bends at sites approximately one and four helix turns from the dyad axis. We have developed two photochemical methods to study the structure of DNA, and in particular that wrapped around the nucleosome core. One method depends on the sensitization of singlet oxygen production by an eosin analogue. We have monitored the rate at which excited state oxygen diffuses into contact with DNA base planes, and find that it attacks the nucleosome with high specificity. We have also mapped the DNA binding of the intercalating dye methylene blue, and conclude that it binds to the same sites accessible to oxygen by diffusion. On the basis of these results we suggest that the DNA in the nucleosome is bent or kinked at two sites, 1.5 helix turns from the dyad axis.

Animals↗
PubMedDate not supplied

Importance of DNA stiffness in protein-DNA binding specificity.

From the first high-resolution structure of a repressor bound specifically to its DNA recognition sequence it has been shown that the phage 434 repressor protein binds as a dimer to the helix. Tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. The centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there. This observation might be explained should the structure of the intervening DNA segment vary with its sequence, or if DNA at the centre of the operator resists the torsional and bending deformation necessary for complex formation in a sequence dependent fashion. We have considered the second hypothesis by demonstrating that DNA stiffness is sequence dependent. A method is formulated for calculating the stiffness of any particular DNA sequence, and we show that this predicted relationship between sequence and stiffness can explain the repressor binding data in a quantitative manner. We propose that the elastic properties of DNA may be of general importance to an understanding of protein-DNA binding specificity.

Base Sequence↗