Effects of Extraction of the H-Subunit from Rhodobacter sphaeroides Reaction Centers on Relaxation Processes Associated with Charge Separation

Effects of extraction of the H-subunit from Rhodobacter sphaeroides photosynthetic reaction centers (RC) on the characteristics of the photoinduced conformational transition associated with electron transfer between photoactive bacterio-chlorophyll and primary quinone acceptor were studied. Extraction of the H-subunit (i.e., the subunit that is not directly bound to electron transfer cofactors) was found to have a significant effect on the dynamic properties of the protein–pigment complex of the RC, the effect being mediated by modification of parameters of the relaxation processes associated with charge separation.     

The dipyridamole effect on the photoactive bacteriochlorophyll interaction with quinone acceptors in reaction centers of purple bacteria

The effect of Dipyridamole (10(-6)-10(-3) M) on the photomobilized electron transport in the system of quinone acceptors Q(A)-Q(B) of isolated photosynthetic reaction centers of Rhodobacter sphaeroides and on its temporary stabilization on Q(B) was studied. Depending on the type of the detergent present in the reaction center (lauryl dimethylamine oxide, Triton X-100, sodium dodecyl sulfate, and sodium cholate), dipyridamole could increase the time of the electron transfer to Q(B). The dipyridamole effect on the efficiency of the electron stabilization on Q(B) for reaction centers with different detergents was revealed in slowing down the process of dark reduction of photoactive bacteriochlorophyll from Q(B) at initial concentrations of added dipyridamole (10(-6)-10(-5) M) with following acceleration of the process at the dipyridamole concentrations of 10(-4)-10(-3) M. The pH lowering from 6.8-7.0 to 5.9-6.0 increased the dipyridamole effect. The possibility of the dipyridamole effect on the structural-dynamic state of the reaction center complex, including its hydrogen bond system, which influences the studied parameters of functional activity, is suggested.     

Monolayers of a model anesthetic-binding membrane protein: formation, characterization, and halothane-binding affinity

hbAP0 is a model membrane protein designed to possess an anesthetic-binding cavity in its hydrophilic domain and a cation channel in its hydrophobic domain. Grazing incidence x-ray diffraction shows that hbAP0 forms four-helix bundles that are vectorially oriented within Langmuir monolayers at the air-water interface. Single monolayers of hbAP0 on alkylated solid substrates would provide an optimal system for detailed structural and dynamical studies of anesthetic-peptide interaction via x-ray and neutron scattering and polarized spectroscopic techniques. Langmuir-Blodgett and Langmuir-Schaeffer deposition and self-assembly techniques were used to form single monolayer films of the vectorially oriented peptide hbAP0 via both chemisorption and physisorption onto suitably alkylated solid substrates. The films were characterized by ultraviolet absorption, ellipsometry, circular dichroism, and polarized Fourier transform infrared spectroscopy. The alpha-helical secondary structure of the peptide was retained in the films. Under certain conditions, the average orientation of the helical axis was inclined relative to the plane of the substrate, approaching perpendicular in some cases. The halothane-binding affinity of the vectorially oriented hbAP0 peptide in the single monolayers, with the volatile anesthetic introduced into the moist vapor environment of the monolayer, was found to be similar to that for the detergent-solubilized peptide.     

Temporary Stabilization of Electron on Quinone Acceptor Side of Reaction Centers from the Bacterium Rhodobacter sphaeroides Wild Type and Mutant SA(L223) Depending on Duration of Light Activation

The dark reduction of photooxidized bacteriochlorophyll (P+) by photoreduced secondary quinone acceptor (QB-) in isolated reaction centers (RC) from the bacterium Rhodobacter sphaeroides wild type and mutant strain SA(L223) depending on the duration of light activation of RC was studied. The kinetics of the dark reduction of P+ decreased with increasing light duration, which is probably due to conformational changes occurring under prolonged light activation in RC from the wild type bacterium. In RC from bacteria of the mutant strain in which protonatable amino acid Ser L223 near QB is substituted by Ala, the dependence of reduction kinetics of P+ on duration of light was not observed. Such dependence, however, became observable after addition of cryoprotectors, namely glycerol and dimethylsulfoxide, to the RC samples from the mutant strain. It was concluded that substitution of Ser L223 with Ala disturbs the native mechanism of electrostatic stabilization of the electron in the RC quinone acceptor site. At the same time, an additional modification of RC hydrogen bonds by glycerol and dimethylsulfoxide probably includes various possibilities for more effective time delay of the electron on QB.     

A model membrane protein for binding volatile anesthetics

Earlier work demonstrated that a water-soluble four-helix bundle protein designed with a cavity in its nonpolar core is capable of binding the volatile anesthetic halothane with near-physiological affinity (0.7 mM Kd). To create a more relevant, model membrane protein receptor for studying the physicochemical specificity of anesthetic binding, we have synthesized a new protein that builds on the anesthetic-binding, hydrophilic four-helix bundle and incorporates a hydrophobic domain capable of ion-channel activity, resulting in an amphiphilic four-helix bundle that forms stable monolayers at the air/water interface. The affinity of the cavity within the core of the bundle for volatile anesthetic binding is decreased by a factor of 4-3.1 mM Kd as compared to its water-soluble counterpart. Nevertheless, the absence of the cavity within the otherwise identical amphiphilic peptide significantly decreases its affinity for halothane similar to its water-soluble counterpart. Specular x-ray reflectivity shows that the amphiphilic protein orients vectorially in Langmuir monolayers at higher surface pressure with its long axis perpendicular to the interface, and that it possesses a length consistent with its design. This provides a successful starting template for probing the nature of the anesthetic-peptide interaction, as well as a potential model system in structure/function correlation for understanding the anesthetic binding mechanism.     

Effect of dipyridamole on recombination of photooxidized bacteriochlorophylla and photoreduced primary quinone in reactive centers of purple bacteria and degradation of form M412 of bacteriorhodopsin

It is shown that the addition of dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4d]py rim idine) (up to 10(-4) M) leads to a drastic acceleration of the dark recombination reaction between photooxidized bacteriochlorophyll and photoreduced primary quinone in reaction centers of Rhodobacter sphaeroides. The value of the acceleration is similar to that registered under cryogenic temperatures. The extent of the effect of dipyridamole derivatives depended on their structure. In wild-type bacteriorhodopsin and D96N mutant, dipyridamole slowed down the Schiff base reprotonation (the kinetics of M412 form decay was registered). It is suggested that dipyridamole can influence the structural and dynamic state of membrane proteins by affecting the system of their hydrogen-bonds and thus modify electron and proton transport processes.     

Impact of methionine oxidation as an initial event on the pathway of human prion protein conversion

Prion diseases comprise a group of fatal neurodegenerative disorders characterized by the autocatalytic conversion of the cellular prion protein PrP^C into the infectious misfolded isoform PrP^Sc. Increasing evidence supports a specific role of oxidative stress in the onset of pathogenesis. Although the associated molecular mechanisms remain to be elucidated in detail, several studies currently suggest that methionine oxidation already detected in misfolded PrP^Sc destabilizes the native PrP fold as an early event in the conversion pathway. To obtain more insights about the specific impact of surface-exposed methionine residues on the oxidative-induced conversion of human PrP we designed, produced, and comparatively investigated two new pseudosulfoxidation mutants of human PrP 121–231 that comprises the well-folded C-terminal domain. Applying circular dichroism spectroscopy and dynamic light scattering techniques we showed that pseudosulfoxidation of all surface exposed Met residues formed a monomeric molten globule-like species with striking similarities to misfolding intermediates recently reported by other groups. However, individual pseudosulfoxidation at the polymorphic M129 site did not significantly contribute to the structural destabilization. Further metal-induced oxidation of the partly unfolded pseudosulfoxidation mutant resulted in the formation of an oligomeric state that shares a comparable size and stability with PrP oligomers detected after the application of different other triggers for structural conversion, indicating a generic misfolding pathway of PrP. The obtained results highlight the specific importance of methionine oxidation at surface exposed residues for PrP misfolding, strongly supporting the hypothesis that increased oxidative stress could be one causative event for sporadic prion diseases and other neurodegenerative disorders.     

Redox-dependent changes in molecular properties of mitochondrial apoptosis-inducing factor

Mitochondrial apoptosis-inducing factor (AIF) is a central player in the caspase-independent cell death pathway whose normal physiological function remains unclear. Our study showed that naturally folded mouse AIF very slowly reacts with NAD(P)H (k cat of 0.2-0.01 s(-1)) forming tight, dimeric, and air-stable FADH2-NAD(P) charge-transfer complexes ineffective in electron transfer. FAD reduction is accompanied by a conformational change involving AIF-specific N-terminal and regulatory 509-559 peptides and the active site His 453, and it affects susceptibility of AIF to calpain and AIF-DNA interaction, the two events critical for initiating caspase-independent apoptosis. Based on our results, we propose that formation of long lived complexes with NAD(P)H and redox reorganization may be functionally important and enable AIF to act as a redox-signaling molecule linking NAD(P)H-dependent metabolic pathways to apoptosis.