The bacteriorhodopsin proton pump: effect of crosslinkings of lysine residues

All six available lysine residues in bacteriorhodopsin were amidinated with dimethyl-3,3'-dithiobispropionimidate, which is a crosslinking agent. The photocycle was studied by measuring light absorption and electric signals. The data show an essential change in the photocycle: instead of single components, the rise of the signal due to the M intermediate can be decomposed into two components, and the decay into three. The life-times and the intensities of these components and in general the proton pumping activity of bacteriorhodopsin depend only negligibly upon pH. Changes upon removing the crosslinks are not significantly different from those in the crosslinked samples. The lysine residues therefore may not be considered of primary importance in proton translocation.     

Angle of the retinal of bacteriorhodopsin in blue membrane

The electric dichroism of purple and cation-depleted (blue) membrane was measured in a.c. electric fields at saturation. A decrease of 5.5° in the direction of the chromophore transition moment with respect to the membrane normal was found upon removal of cations from purple membrane.     

Reversible inhibition of proton release activity and the anesthetic-induced acid-base equilibrium between the 480 and 570 nm forms of bacteriorhodopsin.

In purple membrane added with general anesthetics, there exists an acid-base equilibrium between two spectral forms of the pigment: bR570 and bR480 (apparent pKa = 7.3). As the purple 570 nm bacteriorhodopsin is reversibly transformed into its red 480 nm form, the proton pumping capability of the pigment reversibly decreases, as indicated by transient proton release measurements and proton translocation action spectra of mixture of both spectral forms. It happens in spite of a complete photochemical activity in bR480 that is mostly characterized by fast deprotonation and slow reprotonation steps and which, under continuous illumination, bleaches with a yield comparable to that of bR570. This modified photochemical activity has a correlated specific photoelectrical counterpart: a faster proton extrusion current and a slower reprotonation current. The relative areas of all photocurrent phases are reduced in bR480, most likely because its photochemistry is accompanied by charge movements for shorter distances than in the native pigment, reflecting a reversible inhibition of the pumping activity.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Perturbation of DPPC bilayers by high concentrations of pulmonary surfactant protein SP-B

Deuterium (²H) NMR has been used to observe perturbation of dipalmitoylphosphatidylcholine (DPPC) bilayers by the pulmonary surfactant protein B (SP-B) at concentrations up to 17% (w/w). Previous ²H NMR studies of DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3) bilayers containing up to 11% (w/w) SP-B and DPPC bilayers containing up to 11% (w/w) synthetic SP-B indicated a slight effect on bilayer chain order and a more substantial effect on motions that contribute to decay of quadrupole echoes obtained from bilayers of deuterated DPPC. This is consistent with the perturbation of headgroup-deuterated DPPC reported here for bilayers containing 6 and 9% (w/w) SP-B. For the higher concentrations of SP-B investigated in the present work, ²H NMR spectra of DPPC deuterated in both the headgroup and chain display a prominent narrow component consistent with fast, large amplitude reorientation of some labeled lipid. Similar spectral perturbations have been reported for bilayers in the presence of the antibiotic polypeptide nisin. The observation of large amplitude lipid reorientation at high SP-B concentration could indicate that SP-B can induce regions of high bilayer curvature and thus provides some insight into local interaction of SP-B with DPPC. Such local interactions may be relevant to the formation, in vitro and in vivo, of tubular myelin, a unique structure found in extracellular pulmonary surfactant, and to the delivery of surfactant material to films at the air–water interface.Abbreviations DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol - DPPC-d₆₂ 1,2-perdeuterodipalmitoyl-sn-glycero-3-phosphocholine - DPPC-d₄ 1,2-dipalmitoyl-sn-glycero-3-phospho-(, perdeutero)-choline     

Lipid-induced conformational switch in the membrane binding domain of CTP:phosphocholine cytidylyltransferase: a circular dichroism study

CTP:phosphocholine cytidylyltranferase (CCT) regulates phosphatidylcholine (PC) biosynthesis. Its activity is controlled by reversible interactions with membrane lipids, mediated by an internal segment referred to as domain M. Although domain M peptides adopt an amphipathic alpha-helical structure when membrane bound, the structure of this domain in the context of the whole enzyme in the lipid-free and lipid-bound state is unknown. Here we derive lipid-induced secondary structural changes in CCTalpha using circular dichroism and three deconvolution programs. The analysis of two fragments, CCT236 (CCT1-236, housing the catalytic domain) and a synthetic domain M peptide (CCT237-293) aided the assignment of structural change to specific domains. To carry out this study, we developed a micellar lipid activating system that would avoid generation of CCT-induced lipid vesicle aggregates that interfere with the CD analysis. Lysophosphatidylcholine/phosphatidylglycerol (LPC/PG) mixed micelles supported full activation of CCT and caused an increase in the alpha-helix content of full-length CCT from 25 to 41%, at the expense of all other conformations. LPC/PG also induced an increase in alpha-helix content of the domain M peptide from 7 to 85% at the expense of all other conformers. This lipid system did not significantly affect the secondary structure of CCT236, nor did it affect the proteolytic fragmentation pattern of this region within full-length CCT, suggesting that the region containing the catalytic domain changes very little upon membrane activation of CCT. Our data suggest that lipids trigger a conformational switch in domain M from a mixed structure to an alpha-helix, thus creating a hydrophobic face for membrane insertion. Our results negate the idea that domain M is entirely helical in both the soluble and membrane-bound forms of CCT.     

Decomposition of old organic matter as a result of deeper active layers in a snow depth manipulation experiment

A snow addition experiment in moist acidic tussock tundra at Toolik Lake, Alaska, increased winter snow depths 2–3 m, and resulted in a doubling of the summer active layer depth. We used radiocarbon (?¹⁴C) to (1) determine the age of C respired in the deep soils under control and deepened active layer conditions (deep snow drifts), and (2) to determine the impact of increased snow and permafrost thawing on surface CO₂ efflux by partitioning respiration into autotrophic and heterotrophic components. ?¹⁴C signatures of surface respiration were higher in the deep snow areas, reflecting a decrease in the proportion of autotrophic respiration. The radiocarbon age of soil pore CO₂ sampled near the maximum mid-July thaw depth was approximately 1,000 years in deep snow treatment plots (45–55 cm thaw depth), while CO₂ from the ambient snow areas was ~100 years old (30-cm thaw depth). Heterotrophic respiration ?¹⁴C signatures from incubations were similar between the two snow depths for the organic horizon and were extremely variable in the mineral horizon, resulting in no significant differences between treatments in either month. Radiocarbon ages of heterotrophically respired C ranged from <50 to 235 years BP in July mineral soil samples and from 1,525 to 8,300 years BP in August samples, suggesting that old soil C in permafrost soils may be metabolized upon thawing. In the surface fluxes, this old C signal is obscured by the organic horizon fluxes, which are significantly higher. Our results indicate that, as permafrost in tussock tundra ecosystems of arctic Alaska thaws, carbon buried up to several thousands of years ago will become an active component of the carbon cycle, potentially accelerating the rise of CO₂ in the atmosphere.     

Ionic interactions at both inter-ring contact sites of GroEL are involved in transmission of the allosteric signal: a time-resolved infrared difference study

The biological activity of the double-ring chaperonin GroEL is regulated by complex allosteric interactions, which include positive intra-ring and negative inter-ring cooperativity. To further characterize inter-ring communication, the nucleotide-induced absorbance changes in the vibrational spectrum of the chaperonin GroEL, of two single-point mutants suppressing one inter-ring ionic contact (E461K and E434K) and of a single-ring version of this protein, were investigated by time-resolved infrared difference spectroscopy. Interaction of the nucleotide with the proteins was triggered by its photochemical release from a biologically inactive caged precursor [P3-1-(2-nitro) phenylethyl nucleotide]. The results indicate that (1) ATP binding to the protein induces a conformational change that affects concomitantly both intra-ring and inter-ring communication, and (2) the experimental absorbance changes are sensitive to the double-ring structure of the protein. The characterization of the single-point, inter-ring mutants demonstrates that ionic interactions at both contact sites are involved in the transmission of the allosteric signal. However, both mutations have different effects on the inter-ring interface. While that of E461K still retains ionic contacts sensitive to ATP binding, E434K shows spectroscopic features similar to those of the single-ring version of the protein, therefore suggesting that electrostatic interactions at these contact sites contribute differently to the stability of the inter-ring interface.     

Excitation of the L intermediate of bacteriorhodopsin: electric responses to test x-ray structures

The L intermediate of bacteriorhodopsin was excited, and its electrical response was measured. Two positive components were found in it with respect to the direction of proton pumping: an unresolved fast component, and a slower one (tau=7 micros) of small amplitude. The fast component was assigned to a charge motion corresponding to reisomerization of the retinal moiety, whereas the slow one was attributed to charge rearrangements reestablishing the ground state. Because three x-ray crystallographic structures have recently been reported for the L intermediate, it seemed important to calculate the intramolecular dipole moment changes associated to bR-->L for all three structures, so as to compare them with similar quantities determined from the electrical signals. The results are discussed in terms of amino acid side chains possibly contributing to the observed effect. We propose to use electrical signals as a verification tool for intermediate structures of the photocycle, and thus for molecular models of proton pumping.     

The effect of carbon dioxide enrichment on apparent stem respiration from <Emphasis Type="Italic">Pinus taeda</Emphasis> L. is confounded by high levels of soil carbon dioxide

Huey and Slatkin’s (Q Rev Biol 51:363–384, 1976) cost–benefit model of lizard thermoregulation predicts variation in thermoregulatory strategies (from active thermoregulation to thermoconformity) with respect to the costs and benefits of the thermoregulatory behaviour and the thermal quality of the environment. Although this framework has been widely employed in correlative field studies, experimental tests aiming to evaluate the model are scarce. We conducted laboratory experiments to see whether the common lizard Zootoca vivipara, an active and effective thermoregulator in the field, can alter its thermoregulatory behaviour in response to differences in perceived predation risk and food supply in a constant thermal environment. Predation risk and food supply were represented by chemical cues of a sympatric snake predator and the lizards’ food in the laboratory, respectively. We also compared males and postpartum females, which have different preferred or “target” body temperatures. Both sexes thermoregulated actively in all treatments. We detected sex-specific differences in the way lizards adjusted their accuracy of thermoregulation to the treatments: males were less accurate in the predation treatment, while no such effects were detected in females. Neither sex reacted to the food treatment. With regard to the two main types of thermoregulatory behaviour (activity and microhabitat selection), the treatments had no significant effects. However, postpartum females were more active than males in all treatments. Our results further stress that increasing physiological performance by active thermoregulation has high priority in lizard behaviour, but also shows that lizards can indeed shift their accuracy of thermoregulation in response to costs with possible immediate negative fitness effects (i.e. predation-caused mortality).