Binding of the Tet repressor to nonspecific and specific DNA leads to quenching of the Tet fluorescence by approximately 22% and approximately 35%, respectively. This effect is used for a direct, quantitative characterization of the binding equilibria and dynamics involved in the recognition of the operator by its repressor. From the dependence of the nonspecific binding constant on the ion concentration, it is concluded that nonspecific binding is almost completely driven by the entropy change resulting from the release of three to four Na+ ions from the double helix upon protein binding. Formation of the specific complex is driven by a higher entropy term resulting from the release of seven to eight Na+ ions and in addition by a free energy term of -33 kJ/mol from nonelectrostatic interactions, which are attributed to the specific contacts. The dynamics of the repressor-operator recognition are resolved by stopped-flow measurements at various salt concentrations and for different DNA chain lengths into two separate steps. The first step follows a second-order mechanism and results in an intermediate complex associated with formation of about three to four electrostatic contacts between protein and DNA; apparently, this complex is equivalent to the nonspecific complex. The existence of an intermediate is also indicated by experiments in mixed Na+-Mg2+ buffers, which can be described with high accuracy by competition of Mg2+ and protein. The intermediate complex is formed at a rate of 3 X 10(8) M-1 s-1 and is converted in the second reaction step to the specific complex with a rate constant of 6 X 10(4) s-1, which is almost independent of the salt concentration. Our interpretation and the parameters obtained from our model are confirmed by competition of nonspecific DNA with operator DNA for repressor binding. The observed maximal rate constant of 3 X 10(8) M-1 s-1 is very close to theoretical predictions for the association without a sliding mechanism. The very small dependence of the observed rate constants on the chain length shows that the Tet repressor is not able to slide over any substantial distance even at low salt concentrations. The question of a potential contribution from sliding under our experimental conditions is critically discussed. The absence of sliding in the case of the Tet repressor under physiological conditions is compared with the high sliding efficiency of the lac repressor and is discussed with respect to possible molecular mechanisms of sliding in relation to biological function.

A series of computer simulations of gel patterns assuming non-cooperative binding of a protein to two targets on the same DNA fragment was performed and applied to interprete gel mobility shift experiments of Tet repressor-tet operator binding. While a high binding affinity leads to the expected distribution of free DNA, DNA bound by one repressor dimer and DNA bound by two repressor dimers, a lower affinity or an increased electrophoresis time results in the loss of the band corresponding to the singly occupied complex. The doubly occupied complex remains stable under these conditions. This phenomenon is typical for protein binding to DNA fragments with two identical sites. It results from statistical disproportionation of the singly occupied complex in the gel. The lack of the singly occupied complex is commonly taken to indicate cooperative binding, however, our analysis shows clearly, that cooperativity is not needed to interprete these results. Tet repressor proteins and small DNA fragments with two tet operator sites have been prepared from four classes of tetracycline resistance determinants. The results of gel mobility shift analyses of various complexes of these compounds confirm the predictions. Furthermore, calculated gel patterns assuming different gel mobilities of the two singly occupied complexes show discrete bands only if the electrophoresis time is shorter than the inverse of the microscopic dissociation rate constant. Simulations assuming increasing dissociation rates predict that the two bands first merge into one, which then disappears. This behavior was verified by gel mobility analyses of Tet repressor-tet operator titrations at increased salt concentrations as well as by direct footprinting of the complexes in the gel. It is concluded that comparison of the intensities of the single and the double occupation bands allow a rough estimation of the dissociation rate constant. On this basis the sixteen possible Tet repressor-tet operator combinations can be ordered with decreasing binding affinities by a simple gel shift experiment. The implications of these results for gel mobility analyses of other protein-DNA complexes are discussed.

Beam hardening is a well-known phenomenon for therapeutic accelerator beams passing through matter in narrow beam geometry. This study assesses quantitatively the magnitude of beam hardening of therapeutic beams in water. A formal concept of beam hardening is proposed which is based on the decrease of the mean attenuation coefficient with depth. On the basis of this concept calculations of beam hardening effects are easily performed by means of a commercial spreadsheet program. Published accelerator spectra and the tabulated values of attenuation coefficients serve as input for these calculations. It is shown that the mean attenuation coefficient starts at depth zero with an almost linear decrease and then slowly levels off to a limit value. A similar behavior is found for the beam hardening coefficient. A physically reasonable, semianalytical model is given which fits the data better than previously published functions. The energy dependence of the initial attenuation coefficient is evaluated and shown. It fits well to published experimental data. The initial beam hardening coefficient, however, shows no energy dependence. Its mean value (eta0) approximately 0.006 cm(-1)) is also in close agreement to the measured data.

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The adsorption of bacteriorhodopsin(bR)-containing purple membranes (PM) to black lipid membranes (BLM) was used to study the charge translocation kinetics of bR upon flash excitation.The discharge of the PM-BLM system after charging upon illumination is found to proceed quite slowly (discharge time up to several minutes) but is considerably accelerated by addition of the protonophore FCCP.Therefore, the dependence of the proton transfer kinetics in bR on electrical potentials generated by preceding flashes of varying repetition rate and intensity was investigated. The kinetics are slowed down with increasing flash intensity as well as repetition rate. This effect is partly abolished by small amounts of FCCP.A new model is introduced which takes into account the instantaneous feedback of the electrical potential on the kinetics of the pump current. It explains the observed deviations from first-order kinetics and renders an approach with "distributed kinetics" unnecessary.