Risk factors for malaria: a microepidemiological study in a village in Sri Lanka.

Environmental and socioeconomic risk factors for malaria were studied in a village in Sri Lanka. Over a period of one year, all 49 households in the village were visited every alternate day to obtain information on malaria episodes. Information on risk factors was obtained through questionnaires and direct observations. Age below 17 years (relative risk [RR] = 1.66, 95% confidence interval [95% CI] 1.18-2.35), use of bed nets (RR = 0.16, 95% CI 0.05-0.45) and traditional fumigants (RR = 0.58, 95% CI 0.37-0.93) were independent predictors of malaria. People using anti-mosquito pyrethrum coils had a higher risk for malaria than people living in houses where they were not used (RR = 1.46, 95% CI 1.03-2.07). The build-up of Anopheles culicifacies populations before the start of the transmission season had taken place in a stream near the village. Living close to the stream was a risk factor for malaria early in the transmission season, although this did not reach statistical significance (comparing < 250 m with > 500 m, RR = 2.13, 95% CI 0.96-4.71).

Mechanics of solute translocation catalyzed by enzyme IImtl of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The kinetics of binding of mannitol to enzyme IImtl embedded in the membrane of vesicles with an inside-out or a right-side-out orientation were analyzed at 4 degrees C in the absence of the phosphoryl group donor, P-HPr. The binding to the right-side-out oriented vesicles equilibrated too fast to be monitored by the flow dialysis technique. On the other hand, with the inside-out oriented membrane vesicles two conformational changes of the enzyme could be detected kinetically. One change involved a recruitment of binding sites from a state of the enzyme where the binding sites were inaccessible from the cytoplasmic volume. The second change involved a conformational change of the enzyme that followed upon the initial binding to the cytoplasmic-facing binding site leading to a state with a higher affinity for mannitol. Equilibrium binding to the inside-out and right-side-out oriented membrane vesicles at 4 degrees C indicated that the two transitions did not represent the translocation of the binding site, free and with mannitol bound to it, to the other side of the membrane. Instead, a model is proposed in which the conformational changes represent transitions from states with the binding pocket opened to the cytoplasmic side of the membrane to occluded states of the enzyme in which the binding sites, with or without mannitol bound, are not accessible to either side of the membrane.

Membrane proteins and impure detergents: procedures to purify membrane proteins to a degree suitable for tryptophan fluorescence spectroscopy.

Investigation of the membrane-embedded mannitol permease of Escherichia coli (EIImtl) steady-state tryptophan fluorescence was hampered by fluorescent impurities arising from detergents and other sources during the isolation. The signals from these impurities could not be distinguished from tryptophan fluorescence on the basis of lifetimes or emission spectra. Consequently, a tryptophan-minus mutant of EIImtl, EIImtl(Trp-), was constructed to address this problem. The findings were that the fluorescent impurities, present in the detergents and/or arising from the action of the detergents on plastic vials and tubing used during the isolation procedure, accumulate in enzyme solutions to levels comparable to the signal from the tryptophan residues in the protein. The high affinity of these impurities for EIImtl makes them impossible to remove by dialysis, by reconstitution of the protein with pure phospholipids, or by detergent exchange when the protein is immobilized on a resin. A procedure was developed to completely remove all fluorescent impurities from the nonionic polyethylene glycol-based detergent, decylpenta(ethylene glycol) (C10E5). This detergent and modified isolation procedures yield EIImtl(Trp-) and single tryptophan mutants in which the impurities no longer interfere with the tryptophan emission signal. The methodologies presented in this paper might make it possible to study the fluorescence of tryptophan residues in other membrane proteins without the interference of impurities with similar fluorescence properties.

Mechanistic coupling of transport and phosphorylation activity by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.

Mannitol bound to enzyme IImtl could be trapped specifically by rapid phosphorylation with P-HPr. The assay was used to demonstrate transport of mannitol across the cytoplasmic membrane with and without phosphorylation of mannitol. The latter was 2-3 orders of magnitude slower. The fraction of bound mannitol molecules that was actually phosphorylated, the efficiency of the trap, was less than 50%. The efficiency was not very different for enzyme IImtl embedded in the membrane of vesicles with an inside-out orientation or solubilized in detergent. Subsequently, it is argued that the fraction of the bound mannitol molecules that was not phosphorylated dissociated into the cytoplasmic space. A model for the catalytic mechanism of enzyme IImtl is proposed on the basis of interpretations of the present experiments. The main features of the model are the following: (i) mechanistically, the coupling between transport and phosphorylation is less than 50%; (ii) in the physiological steady state of mannitol transport and metabolism, the coupling is 100%; (iii) phosphorylated enzyme IImtl catalyzes facilitated diffusion at a high rate; (iv) the state of phosphorylation of the cytoplasmic domain modulates the activity of the translocator domain; (v) the enzyme catalyzes phosphorylation of free cytoplasmic mannitol at least as fast as it catalyzes transport plus phosphorylation of free periplasmic mannitol.

Interaction between the cytoplasmic and membrane-bound domains of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.

Sulfhydryl reagents affected the binding properties of the translocator domain, NIII, of enzyme IImtl in two ways: (i) the affinity for mannitol was reduced, and (ii) the exchange rate of bound and free mannitol was increased. The effect on the affinity was very much reduced after solubilization of enzyme IImtl in the detergent decylPEG. The effects were caused exclusively by reaction of the sulfhydryl reagents with the cysteine residue at position 384 in the primary sequence. Interaction between two domains is involved, since Cys384 is located in the cytoplasmic domain, CII. When Cys384 was mutated to serine, the enzyme exhibited the same binding properties as the chemically modified enzyme. The data support our proposal that phosphorylation of enzyme IImtl drastically reduces the activation energy for the translocation step through interaction between domains CII and NIII [Lolkema J. S., ten Hoeve-Duurkens, R. H., Swaving Dijkstra, D., & Robillard, G. T. (1991) Biochemistry (preceding paper in this issue)]. Functional interaction between the translocator domain, NIII, and domain CI was investigated by phosphorylation of His554, located in domain CI, in the C384S mutant. No effect on the binding properties was observed. In addition, the binding properties were insensitive to the presence of the soluble phosphotransferase components enzyme I and HPr.

Dynamic fluorescence spectroscopy on single tryptophan mutants of EII(mtl) in detergent micelles. Effects of substrate binding and phosphorylation on the fluorescence and anisotropy decay.

The effects of substrate and substrate analogue binding and phosphorylation on the conformational dynamics of the mannitol permease of Escherichia coli were investigated, using time-resolved fluorescence spectroscopy on mutants containing five single tryptophans situated in the membrane-embedded C domain of the enzyme [Swaving Dijkstra et al. (1996) Biochemistry 35, 6628-6634]. Since no fluorescent impurities are present in these mutants, the changes in fluorescence and anisotropy could be related with changes in the tryptophan microenvironment. Tryptophans at positions 30 and 42 showed changes in fluorescence intensity decay upon binding mannitol, which were reflected in the changes in lifetime distribution patterns. The disappearance of the shortest-lived decay component in these mutants, as well as in the mutant with a single tryptophan at position 109, indicates a change in the local environment such that quenching via neighboring side chains or solvent is reduced. Phosphorylation at histidine 554 and cysteine 384, located in the cytoplasmatic A and B domains of EII(mtl), respectively, induced an increase in the average fluorescence lifetimes of all of the tryptophans. The effect was most pronounced for tryptophans 30 and 109 which show large increases in the average fluorescence lifetime mainly due to loss of short-lived decay components. A correlation time distribution of the individual tryptophans deduced from an analysis of the anisotropy decay showed that they differed in their rotational mobility with tryptophan 30 showing the least local flexibility. Phosphorylation resulted in immobilization of W109 which, together with changes in the average fluorescence lifetime, is evidence for a conformational coupling between the phosphorylated B domain and the C domain. The influence of mannitol binding on the rotational behavior of the tryptophans is limited; it induces more internal flexibility at all tryptophan positions. A rotational correlation time of 30 ns was resolved for tryptophan 30, which probably represents a rotational mode of the micelle-embedded C-domain of EII(mtl) or a portion thereof. Upon phosphorylation, this rotational correlation time increases to 50 ns, probably reflecting a changed spatial orientation of W30 with respect to the C domain. Although kinetic experiments have shown that none of the tryptophans is essential for the catalytic activity of EII(mtl), it is significant that the residues most sensitive to mannitol binding, W30 and W42, are both located in the first membrane-spanning alpha-helix, a portion of which is highly conserved among mannitol-specific EII's of different bacteria.

The membrane-bound domain of the phosphotransferase enzyme IImtl of Escherichia coli constitutes a mannitol translocating unit.

The orientation of the mannitol binding site on the Escherichia coli phosphotransferase enzyme IImtl in the unphosphorylated state has been investigated by measuring mannitol binding to cytoplasmic membrane vesicles with a right-side-out and inside-out orientation. Enzyme IImtl is shown to catalyze facilitated diffusion of mannitol at a low rate. At equilibrium, bound mannitol is situated at the periplasmic side of the membrane. The apparent binding constant is 40 nM for the intact membranes. Solubilization of the membranes in detergent decreases the affinity by about a factor of 2. Inside-out membrane vesicles, treated with trypsin to remove the C-terminal cytoplasmic domain of enzyme IImtl, showed identical activities. These experiments indicate that the translocation of mannitol is catalyzed by the membrane-bound N-terminal half of enzyme IImtl which is a structurally stable domain.

A fluorescence study of single tryptophan-containing mutants of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent mannitol transport system.

The fluorescence properties of six different single Trp mutants of the mannitol-specific transporter of Escherichia coli were studied in order to derive structural information at different locations in the enzyme. The use of pure detergent and special protein purification protocols was essential for reliable fluorescence spectra, as judged from tyrosine-like fluorescence in a tryptophan-minus mutant (Robillard et al., 1996). The steady-state fluorescence spectra of EIImtl mutants with single tryptophan residues at positions 30, 42, 109, 117, 320, and 384 provided information concerning the polarity of the environment and the effects of mannitol binding at these positions. Tryptophan positions 42, 109, and 117 with emission maxima ranging from 337 to 340 nm are relatively polar, and position 384 with an emission maximum at 346 nm is highly polar, whereas position 30 is highly apolar with a maximum at 324 nm. The fluorescence characteristics of tryptophan 30 suggest a buried position in a hydrophobic part of the enzyme, which is confirmed by the low Stern-Volmer quenching constant for I- quenching. Positions 109 and 117 show the highest quenching constants, indicating the most exposed positions, whereas positions 320 and 42 are moderately quenched, by I-. The tryptophan residue at position 384 is, even in the absence of externally added quencher, very strongly quenched, possibly by the carboxylate from aspartate 384 or by a tyrosinate at position 458 which is nearby in the folded protein (AB et al., in preparation; van Montfort et al., in preparation). The observed emission maxima and accessibilities of the tryptophans at the different positions are consistent with the predicted topology of the enzyme (Sugiyama et al., 1991). When mannitol is bound to wild-type EIImtl, an increase in fluorescence emission intensity was observed (Wood, 1988) which can now be attributed primarily to increased fluorescence intensity of the tryptophan at position 30.