Csilla Gergely

Kinetic and Thermodynamic Study of the Bacteriorhodopsin Photocycle over a Wide pH Range

The photocycle of bacteriorhodopsin and its thermodynamic parameters were studied in the pH range of 4.5-9. Measurements were performed at five different wavelengths (410, 500, 570, 610, and 650 nm), in the time interval 300 ns to 0.5 s, at six temperatures between 5 and 30 degreesC. Data were fitted to different photocycle models. The sequential model with reversible reactions gave a good fit, and the linear character of the Eyring plots was fulfilled. The parallel model with unidirectional reactions gave a poor fit, and the Eyring plot of the rate constants did not follow the expected linear behavior. When a parallel model with reversible reactions, which has twice as many free parameters as the sequential model, was considered, the quality of the fit did not improve and the Eyring plots were not linear. The sequential model was used to determine the thermodynamic activation parameters (activation enthalpy, entropy, and free energy) of the transitions and the free energy levels of the intermediates. pH dependence of the parameters revealed details of the transitions between the intermediates: the transitions M1 to M2 and N to O disclosed a large entropy increase, which could be interpreted as a loosening of the protein structure. The pH dependence of the energy levels explains the disappearance of intermediate O at high pH. A hypothesis is proposed to interpret the relation between the observed pKa of the photocycle energetics and the role of several amino acids in the protein.

Electric Signals during the Bacteriorhodopsin Photocycle, Determined over a Wide pH Range

From the electric signals measured after photoexcitation, the electrogenicity of the photocycle intermediates of bacteriorhodopsin were determined in a pH range of 4.5-9. Current measurements and absorption kinetic signals at five wavelengths were recorded in the time interval from 300 ns to 0.5 s. To fit the data, the model containing sequential intermediates connected by reversible first-order reactions was used. The electrogenicities were calculated from the integral of the current signal, by using the time-dependent concentrations of the intermediates, obtained from the fits. Almost all of the calculated electrogenicities were pH independent, suggesting that the charge motions occur inside the protein. Only the N intermediate exhibited pH-dependent electrogenicity, implying that the protonation of Asp96, from the intracellular part of the protein, is not from a well-determined proton donor. The calculated electrogenicities gave good approximations of all of the details of the measured electric signals.

The effect of azide on the photocycle of bacteriorhodopsin

Abstract Azide has previously been shown to have an effect on the photocycle of bacteriorhodopsin mutants. In appropriate mutants, both the proton release from the Schiff base and reprotonation are accelerated. However, no effect on native bacteriorhodopsin has been found. In this work, we show that protonated azide influences the photocycle of native bacteriorhodopsin, although to a lesser extent than in certain mutants. Several transitions are influenced by protonated azide: however, the overall effect is dominated by the modification of a single step. The M to N transition, i.e. reprotonation of the Schiff base, is accelerated by azide. As a consequence of this kinetic effect, the concentrations of several intermediates are influenced. The interpretation of the kinetic effect of azide involves an alternative parallel reaction path from the M form to the ground state in native bacteriorhodopsin. It is concluded that there are two routes of Schiff base reprotonation: via mediation by Asp-96 (in this case, the molecule returns to the ground state through the N intermediate; this is the generally accepted scenario): via Schiff base reprotonation directly from the external medium (in this case, the molecule returns from another M form to the ground state in one step).

Porous silicon scaffolds for stem cells growth and osteodifferentiation

Abstract: Stem cells constitute the source of differentiated cells for the generation of tissues during development and for regeneration of tissues that are diseased or injured postnatally. Stem cell-based therapy often requires a scaffold to carry cells and/or growth factor to the injured site. Porous silicon (PSi) is a promising biomaterial for tissue engineering, as it is both non-toxic and bioresorbable. Surface modification can offer control over the degradation rate of PSi and can also impart properties to promote cell adhesion. Thus, coupling the good proliferation and differentiation capacities of either adult mesenchymal stem cells (MSC) or embryonic stem cells (ESC) with the textural and chemical properties of the PSi substrates provides an interesting approach for therapeutic use. Moreover, the PSi nanostructure and the release of silicic acid have positive effect on precursor cell osteodifferentiation and mineralized matrix formation.

Orientation of membrane fragments containing (Na+ + K+)-ATPase

Abstract Electro-optical measurements were performed on membrane fragments containing (Na + + K + )-ATPase. Data showed that these disk-shaped membrane fragments have a permanent electric dipole moment. Taking into account the size distribution of the membranes determined by electron microscopy, the value of the dipole moment is (5.5 ± 0.5) × 10 −23 C m for a membrane of 1 μm 2 area. This value does not depend on the conformational state of the (Na + + K + )-ATPase (E 1 and E 2 ), however, if an uncleavable ATP analogue is added to the enzyme a significant increase in the permanent dipole moment is observed. The polarizability of the membrane fragments is less than 10 −29 F m 2 .