Josef F. Holzwarth

Static and dynamic studies of the potential-sensitive membrane probe RH421 in dimyristoylphosphatidylcholine vesicles

The dynamics of the potential-sensitive styryl dye RH421 in dimyristoylphosphatidylcholine vesicles have been investigated above and below the main phase transition temperature using iodine-laser temperature-jump relaxation spectrophotometry and time-resolved fluorescence lifetime measurements. Equilibrium fluorescence titrations have shown that the affinity of the dye for the membrane is much higher in the liquid-crystalline state than in the gel state. The interaction can be described by either a partition or a binding model and a theory is presented providing a relation between these two approaches. In the liquid-crystalline state bound dye exhibits steady-state fluorescence relaxation processes in the submicrosecond and millisecond time range following a temperature jump. Time-resolved fluorescence measurements show a variation in the fluorescence lifetime across the emission spectrum, suggesting an excited-state process occurring on the subnanosecond time scale. These processes are most likely related to dye and/or lipid reorientation following the temperature jump or excitation pulse. Temperature-dependent changes in the fluorescence excitation spectrum of bound dye suggest that the dye exists in at least two different sites within the membrane.

Flexible loops of thread-like micelles are formed upon interaction of l-α-dimyristoyl-phosphatidylcholine with the biosurfactant surfactin as revealed by cryo-electron tomography

Vesicles of L-alpha-dimyristoyl-phosphatidylcholine (DMPC) are known to disintegrate upon treatment with surfactin, a lipoheptapeptide biosurfactant from Bacillus subtilis OKB 105, as was observed by static light scattering (SLS) and cryo-transmission electron microscopy (cryo-TEM) recently. The lysis of DMPC bilayers occurs strongly dependent on the surfactin concentration according to a three-stage model. Unilamellar DMPC vesicles are disrupted to form sheet-like lamellar intermediates at a moderate surfactant concentration, but undergo a transition towards smaller particles of unknown structure at a higher surfactant concentration according to earlier neutron scattering experiments. Here we present direct structural evidence from cryo-electron tomography data that thread-like micelles with a uniform diameter of 6.5 nm are organized into loops of different sizes at a surfactin concentration of > 15 mol%.

Temperature dependent intermediate structures during the main phase transition of dimyristoyl phosphatidylcholine vesicles a combined iodine laser-temperature jump and time resolved cryo-electron microscopy study

The kinetics of the main phase transition of dimyristoylphosphatidyl choline (DMPC) unilamellar vesicles were investigated in the time range from microseconds to seconds. Iodine laser-temperature jump (ILTJ) experiments showed three discrete relaxation phenomena. Time resolved cryo-electron microscopy (CEM) was applied to produce images of intermediate states typical for the relaxation times of lipid vesicles in the micro- to millisecond time window. A careful measurement of the rate of temperature decrease observed during the production of vitrified lamellae of aqueous samples on a copper grid was performed. The best conditions resulted in average rates of cooling of 3 x 10(4) K/s. By comparing the images from CEM of DMPC vesicle samples vitrified above, at, and below the phase transition temperature a structural model was designed, which explains the temperature jump relaxation times in the micro- to millisecond time range by the formation and disappearance of coexisting clusters of crystalline, intermediate, and fluid lipid areas inside the DMPC bilayers.

Influence of allosteric effectors on the kinetics and equilibrium binding of phosphoenolpyruvate (PEP) to phosphoenolpyruvate carboxylase (PEPC) from Zea mays

Phosphoenolpyruvate carboxylase (PEPC) the carbon dioxide processing enzyme of C(4) plants, shows the features of an allosteric enzyme. Allosteric activators such as D-glucose-6-phosphate and glycine increase the affinity of PEPC for its substrate PEP at pH 8.0 and pH 7.0. Allosteric inhibitors like L-malate and L-aspartate predominantly decrease the affinity of the carboxylase for PEP at pH 7.0. This was demonstrated by determination of the enzymatic activity and stopped flow (SF) fluorimetry. The binding reaction of PEP to PEPC from Zea mays was measured using the fluorescence probe 2-p-toluidinonaphthalene-6-sulfonate (TNS). The kinetics are described by an allosteric mechanism with a fast reversible bimolecular binding step of PEP to a high affinity (tensed) form of PEPC, which is in equilibrium with its low affinity (relaxed) form. The influence of allosteric effectors on the conformational transition step is demonstrated in support of the description of the kinetics of PEPC by applying a concerted allosteric mechanism as introduced by Monod, Wyman and Changeux. In summary, we present data for the influence of allosteric activators on the kinetics of PEP binding to PEPC and on the concentration dependence of the isomerisation reaction between two allosteric forms of PEPC.

Microcalorimetric determination of the reaction enthalpy changes associated with the carboxylase and oxygenase reactions catalysed by ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO)

The total reaction enthalpy change ΔrH associated with the carboxylation and oxygenation of D-ribulose 1,5-bisphosphate (RuBP) catalysed by RUBISCO from spinach and Rhodospirillum rubrum was measured for the first time with sensitive microcalorimetry. The enthalpy changes Δr,cH and Δr,oH associated with the carboxylase and oxygenase reactions were determined separately using the known ratio of these reactions. Δr,cH and Δr,oH corrected for the heat of neutralisation due to proton release during the overall carboxylation or oxygenation processes. For the corrected enthalpy change Δr,cH′ of the carboxylase reaction catalysed by RUBISCO of spinach a value of −21.34±0.4 kJ mol−1 was determined. In the case of the oxygenase reaction for RUBISCO of spinach a reaction enthalpy change Δr,oH′ of −319.1±9.6 kJ mol−1 which is typical for energy rich reactions, was found. This value is comparable with Δr,oH′=−291.4±5.8 kJ mol−1 associated with the oxygenase reaction catalysed by Co2+-activated RUBISCO from R. rubrum.