Interaction of internal water molecules with the schiff base in the L intermediate of the bacteriorhodopsin photocycle

In the photocycle of bacteriorhodopsin (BR), the first proton movement, from the Schiff base to Asp85, occurs after the formation of the L intermediate. In L, the C [double bond] N bond of the Schiff base is strained, and the nitrogen interacts strongly with its counterion. The present study seeks to detect the interaction of internal water molecules with the Schiff base in L using difference FTIR spectroscopy at 170 K. The coupled modes of the hydrogen-out-of plane bending vibrations (HOOPs) of the N-H and C(15)-H of the protonated Schiff base are detected as a broad band centered at 911 cm(-1) for BR. A set of bands at 1073, 1064, and 1056 cm(-1) for L is shown to arise from the coupling of the HOOP with the overtones of interacting water O-H vibrations. Interaction with water was shown by the decreased intensity of the HOOPs of L in H(2)(18)O and by the influence of mutants that have been shown to perturb specific internal water molecules in BR. In contrast, the HOOP band of initial BR was not affected by these mutations. In D85N, the coupled HOOP of BR is depleted, while the coupled HOOPs of L are shifted. The results indicate that the Schiff base interacts with water in the L state but in a different manner than in the BR state. Moreover, the effects of mutations suggest that cytoplasmic water close to Thr46 (Wat46) either interacts stronger with the Schiff base in L or that it is important in stabilizing another water that does.     

Chloride binding regulates the Schiff base pK in gecko P521 cone-type visual pigment

The binding of chloride is known to shift the absorption spectrum of most long-wavelength-absorbing cone-type visual pigments roughly 30 nm to the red. We determined that the chloride binding constant for this color shift in the gecko P521 visual pigment is 0.4 mM at pH 6.0. We found an additional effect of chloride on the P521 pigment: the apparent pKa of the Schiff base in P521 is greatly increased as the chloride concentration is increased. The apparent Schiff base pKa shifts from 8.4 for the chloride-free form to >10.4 for the chloride-bound form. We show that this shift is due to chloride binding to the pigment, not to the screening of the membrane surface charges by chloride ions. We also found that at high pH, the absorption maximum of the chloride-free pigment shifts from 495 to 475 nm. We suggest that the chloride-dependent shift of the apparent Schiff base pKa is due to the deprotonation of a residue in the chloride binding site with a pKa of ca. 8.5, roughly that of the Schiff base in the absence of chloride. The deprotonation of this site results in the formation of the 475 nm pigment and a 100-fold decrease in the pigment's ability to bind chloride. Increasing the concentration of chloride results in the stabilization of the protonated state of this residue in the chloride binding site and thus increased chloride binding with an accompanying increase in the Schiff base pK.     

The proton release group of bacteriorhodopsin controls the rate of the final step of its photocycle at low pH

The factors determining the pH dependence of the formation and decay of the O photointermediate of the bacteriorhodopsin (bR) photocycle were investigated in the wild-type (WT) pigment and in the mutants of Glu-194 and Glu-204, key residues of the proton release group (PRG) in bR. We have found that in the WT the rate constant of O --> bR transition decreases 30-fold upon decreasing the pH from 6 to 3 with a pKa of about 4.3. D2O slows the rise and decay of the O intermediate in the WT at pH 3.5 by a factor of 5.5. We suggest that the rate of the O --> bR transition (which reflects the rate of deprotonation of the primary proton acceptor Asp-85) at low pH is controlled by the deprotonation of the PRG. To test this hypothesis, we studied the E194D mutant. We show that the pKa of the PRG in the ground state of the E194D mutant, when Asp-85 is protonated, is increased by 1.2 pK units compared to that of the WT. We found a similar increase in the pKa of the rate constant of the O --> bR transition in E194D. This provides further evidence that the rate of the O --> bR transition is controlled by the PRG. In a further test, the E194Q mutation, which disables the PRG and slows proton release, almost completely eliminates the pH dependence of O decay at pHs below 6. A second phenomenon we investigated was that in the WT at neutral and alkaline pH the fraction of the O intermediate decreases with pKa 7.5. A similar pH dependence is observed in the mutants in which the PRG is disabled, E194Q and E204Q, suggesting that the decrease in the fraction of the O intermediate with pKa ca. 7.5 is not controlled by the PRG. We propose that the group with pKa 7.5 is Asp-96. The slowing of the reprotonation of Asp-96 at high pH is the cause of the decrease in the rate of the N --> O transition, leading to the decrease in the fraction of O.     

Two groups control light-induced schiff base deprotonation and the proton affinity of asp(85) in the Arg(82)His mutant of bacteriorhodopsin

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 &mgr;s to 75 &mgr;s with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.     

Transovarial and transphasic transmissions of Borrelia by the taiga tick Ixodes persulcatus (Ixodidae)

Three generations of the taiga tick Ixodes persulcatus, the descendants from naturally infected females, have been examined by means of dark field and phase contrast microscopies and indirect immunofluorescent reactions with monoclonal antibodies. Location of borreliae in oocytes was examined by means of electron microscopy. The examined ticks derived from 9 females collected in the Novgorod Province, from 6 females of 1st laboratory generation and 5 females of the 2nd generation. In total, 250 larvae, 178 nymphs, 59 females and 70 males of three consequent generation have been examined. Almost 100% of descendants of naturally infected females were infected with Borrelia burgdorferi s. l. and similar infection rate was observed in unfed tick larvae collected in field conditions. The borreliae received transovarially to larvae of the 1st generation then were transmitted to 100% nymphs and imago of this generation and two next generations.     

The role of endothelial mechanosensitivity in vessel bed responses to changes in blood pressure in cats

In 19 anaesthetised cats, the response of vascular bed to increasing perfusion pressure at a constant blood flow perfusion consisted of two phases: a myogenic constriction and a subsequent arterial dilatation. The latter depended on ability of the endothelium to relax the smooth muscle under stress. The findings suggest that the control of the smooth muscle tone by a stress has to fight against the myogenic constriction and thus determines the changes in vascular resistance induced by an increased arterial pressure.     

Effect of chronic stimulation of the negative and positive emotionogenic centers of the hypothalamus on development of cardiac arrhythmias]

Chronic experiments were conducted on 46 rabbits; a study was made of the changes in cardiac activity arising in prolonged (up to 1--2 weeks) electric stimulation of the ventro-median nuclei of the hypothalamus. Disturbances of the cardiac rhythm in the form of tachycardia, atrial flutter and fibrillation, ventricular extrasystole and paroxysmal ventricular tachysystole occurred mostly during the first days of stimulation and ceased after inderal infusion. Functional "weakness" of the cardiac pacemakers was revealed in the animals subjected to 1--2-week stimulation of the ventromedian nuclei. This was indicated by the post-stimulation suppression of the pacemaker automaticity and the appearance of Liciani's periods. Disturbances of the cardiac rhythm originating in stimulation of the ventromedian nuclei ceased in stimulation of the lateral hypothalamic field.     

The characteristics of the ixodid tick-vertebrate animal parasitic system]

The parasitic system ixodid tick (parasite)--vertebrate animal (host) is relatively stable in space and time. Equilibrium state in the system is maintained at the low levels of the hosts' infection and moderate intensity of their immunity. Parasite sensitizes the host's organism at the stage of feeding on antigens of its saliva and the host develops different degrees of resistance preventing the subsequent individuals of ticks from normal feeding. Antitick immunity is species specific. Its intensity is defined by the species belonging of the parasite and host, intensity and intervals between infections, availability of "anti-immune mechanisms" in tick and by many other factors, which are realized at the feeding stage. Regulation of the number of ticks, depending on their abundance in the host's population, is attained due to the oversparse, close to negative binomial distribution on hosts. This mechanism functions on the principle of feedback, so that at the excessive number of the parasite some individuals in the host's population, which are especially subjected to infection, do not cope with parasitic burden and die. However, ticks, which failed to finish their feeding and represent a disproportionately great part of the whole parasite's population, die together with them and the parasitic system quickly restores its stability. In anthropocoenoses and ecosystems at different stages of anthropogenic transformation mutual regulation mechanisms of the parasite and host number break down. As a consequence, extremely high rises in the number of ticks and epizootics of agricultural animals associated with them can occur.     

The N intermediate of bacteriorhodopsin at low temperatures: stabilization and photoconversion

In aqueous suspensions of purple membranes (pH 10.2, 0.4 M KCl) an intermediate having an absorption maximum at 570-575 nm (at -196 degrees C) was produced by first heating the M intermediate up to -30 degrees C and then stabilizing it by subsequent cooling to -60 degrees C. We suggest that this species is the intermediate N (or P or R) found and characterized earlier near room temperature. Upon illumination at -196 degrees C N is transformed into a bathochromically absorbing species KN which has an absorption maximum near 605 nm and an extinction 1.35 times that of N. This light reaction is photoreversible. The quantum yield ratio for the forward and back reaction is 0.18 +/- 0.02. The maximum photo steady state concentration of KN is about 0.24. The N intermediate was also trapped in water suspensions of purple membranes at neutral pH and low salt concentration by illumination at lambda greater than 620 nm during cooling. In addition to N another intermediate absorbing in the red (maximum at 610-620 nm) was accumulated in smaller amounts. It is not photoactive at -196 degrees C and apparently is the O intermediate or a photoproduct of N.