Carbohydrate dynamics at a micellar surface: GD1a headgroup transformations revealed by NMR spectroscopy.

The conformational dynamics of the carbohydrate headgroup of ganglioside GD1a, NeuAc alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer, anchored in a perdeuterated dodecylphosphocholine micelle in aqueous solution, were probed by high resolution NMR spectroscopy. The observed 1H/1H NOE interactions revealed conformational averaging of the terminal NeuAc alpha 2-->3Gal and Gal beta 1-->3GalNAc glycosidic linkages. The pronounced flexibility of this trisaccharide moiety was substantiated further by two-dimensional proton-detected 13C T1, T1 rho and 1H/13C NOE measurements. The anchoring effect of the micelle allowed the detection of conformational fluctuations of the headgroup on the time scale of a few hundred picoseconds. NMR experiments performed on the GD1a/DPC micelles in H2O at low temperatures permitted the observation of hydroxyl proton resonances, contributing valuable conformational information.     

Fourier transform infrared spectroscopy characterization of the lamellar and nonlamellar structures of free lipid A and Re lipopolysaccharides from Salmonella minnesota and Escherichia coli.

The structural polymorphism of free lipid A and deep rough mutant lipopolysaccharide (LPS Re) from Salmonella minnesota strain R595 and Escherichia coli strain F515 was characterized by Fourier transform infrared (IR) spectroscopy. For this, the beta <--> alpha phase states and the three-dimensional supramolecular structures, the latter deduced from small-angle synchrotron radiation x-ray diffraction, were investigated at different water contents, Mg2+ concentrations, and temperatures. The analysis of the IR data for vibrations originating from the hydrophobic moiety shows that the beta <--> alpha acyl chain melting is strongly expressed only for the stretching and scissoring modes of the methylene groups. Vibrational groups originating from the interface region sense the acyl chain melting well (ester carbonyl bands) or only weakly (amide bands), and those resulting from the pure polar moiety not at all. From the x-ray data, the existence of lamellar (L), different cubic, and, for lipid A and LPS R595, also inverted hexagonal (HII) structures could be proven in the temperature range 20-80 degrees C with cubic <--> cubic and cubic <--> HII transitions for the Mg(2+)-free and L <--> HII transitions for the Mg(2+)-containing samples. These structural transitions can be characterized most readily by specific changes of the vibrational bands resulting from the interface region: the ester carbonyl and the amide bands. The magnitude of the changes corresponds to that of the structural rearrangement, i.e., is highest for the L <--> HII, lower for the cubic <--> HII, and lowest for the cubic <--> cubic transitions. The structural transitions are only marginally expressed for vibrational bands of the hydrophobic moiety. Similarly, the band contours of vibrations from the hydrophilic region are no indicators of the structural reorientations except for the carboxylate bands of LPS Re. Particularly the stretching vibrations of the phosphate groups are nearly completely invariant; the absolute values of their half bandwidths, however, differ significantly for lipid A and LPS Re, which seems to be of biological relevance. The ability of IR spectroscopy to detect supramolecular changes also beyond the measurability by x-ray diffraction, i.e., at water contents > 95 to 99.5%, is demonstrated.     

Quaternary structure dynamics and carbon monoxide binding kinetics of hemoglobin valency hybrids.

The kinetics of CO binding and changes in quaternary structure for symmetric valency hybrids of human hemoglobin have been extensively studied by laser photolysis techniques. Both alpha+beta and alpha beta+ hybrids were studied with five different ferric ligands, over a broad range of CO concentrations and photolysis levels. After full CO photolysis, the hybrid tetramers switch extensively and rapidly (< 200 microseconds) to the T quaternary structure. Both R --> T and T --> R transition rates for valency hybrid tetramers with 0 and 1 bound CO have been obtained, as well as the CO association rates for alpha and beta subunits in the R and T states. The results reveal submillisecond R reversible T interconversion, and, for the first time, the changes in quaternary rates and equilibria due to binding a single CO per tetramer have been resolved. The data also show significant alpha-beta differences in quaternary dynamics and equilibria. The allosteric constants do not vary with the spin states of the ferric subunits as predicted by the Perutz stereochemical model. For the alpha beta+CN hybrid the kinetics are heterogeneous and imply partial conversion to a T-like state with very low (seconds) R reversible T interconversion.     

The solution structure of a 3'-phenazinium (Pzn) tethered DNA-RNA duplex with a dangling adenosine: r(5'G-AUUGAA3'):d(5'TCAATC3'-Pzn).

The 3'-Pzn group tethered to an oligo-DNA stabilizes a DNA-RNA hybrid duplex structure by 13 degrees C compared to the natural counterpart. This report constitutes the first full study of the conformational features of a hybrid DNA-RNA duplex, which has been possible because of the unique stabilization of this rather small duplex by the tethered 3'-Pzn moiety (Tm approximately 40 degrees C from NMR). In this study, a total of 252 inter- and intra-strand torsional and distance constraints along with the full NOE relaxation matrix, taking into account the exchange process of imino and amino protons with water, have been used. The 3'-Pzn-promoted stabilization of the DNA-RNA hybrid duplex results in detailed local conformational characteristics such as the torsion angles of the backbone and sugar moieties that are close to the features of the other natural DNA-RNA hybrids (i.e. sugars of the RNA strand are 3'-endo, but the sugars of the DNA strand are intermediate between A- and B-forms of DNA, 72 degrees < P < 180 degrees; note however, that the sugars of our DNA strand have a C1-exo conformation: 131 degrees < P < 154 degrees). This study suggests that 3'-Pzn-tethered smaller oligo-DNA should serve the same purpose as a larger oligo-DNA as a antisense inhibitor of the viral mRNA. Additionally, these types of tethered oligos have been found to be relatively more resistant to the cellular nuclease. Moreover, they are taken up quite readily through the cellular membrane (14) compared to the natural counterparts.     

Effects of hypoxanthine substitution on bleomycin-mediated DNA strand degradation in DNA-RNA hybrids.

We have reported on the differences in site-specific cleavage between DNA and DNA-RNA hybrids by various prototypic DNA cleavers (accompanying paper). In the case of bleomycin (BLM), degradation at 5'-GC-3'sites was suppressed relative to the same sequence in double-stranded DNA, while 5'-GT-3' damage remained constant. We now present results of our further investigation on the chemical and conformational factors that contribute to BLM-mediated DNA strand cleavage of DNA-RNA hybrids. Substitution of guanine by hypoxanthine on the RNA strand of hybrids resulted in a significant enhancement of 5'-GC-3' site damage on the DNA strand relative to double-stranded DNA, thus reversing the suppression noted at these sites. Additionally, 5'-AT-3' sites, which are damaged significantly more in the hybrid than in DNA, exhibit decreased product formation when hypoxanthine is present on the RNA strand of hybrids. However, when hypoxanthine is substituted for guanine on the DNA strand (a GC cleavage site becomes IC), 5'-IT-3' and 5'-IC-3' site cleavage is almost completely suppressed, whereas AT site cleavage is dramatically enhanced. The priority in metallobleomycin site-specific cleavage of hybrids changes with hypoxanthine substitution: the cleavage priority is AT > GT > GC in native hybrid; GC > GT > AT in hybrids substituted with hypoxanthine in the RNA strand; AT >> GT approximately GC in hybrids substituted with hypoxanthine in the DNA strand. The results of kinetic isotope effect studies on BLM cleavage are presented and, in most cases, the values are larger for the hypoxanthine-substituted hybrid. The results suggest that the 2-amino groups of guanine residues on both strands of the nucleic acid play an important role in modulation of the binding and cleavage specificity of BLM.     

Structural studies of lipooligosaccharides from Haemophilus ducreyi ITM 5535, ITM 3147, and a fresh clinical isolate, ACY1: evidence for intrastrain heterogeneity with the production of mutually exclusive sialylated or elongated glycoforms.

The structures of the lipooligosaccharides (LOSs) from Haemophilus ducreyi ITM 5535 and ITM 3147 and a fresh clinical isolate, ACY1, have been investigated. Oligosaccharides were obtained from phenol-water-extracted LOS by mild acid hydrolysis and were studied by methylation analysis, fast atom bombardment and electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. The major oligosaccharide obtained from all strains was a nonasaccharide with the structure beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Galp-(1-->4)-D-a lpha-D-Hepp- (1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp - (1-->3)]4)-L-alpha-D-Hepp-Kdo (Kdo stands for 3-deoxy-D-manno-octulosonic acid) and is thus identical to that identified as the major oligosaccharide in H. ducreyi ITM 2665 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J.A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Electrospray ionization mass spectrometry on O-deacylated LOS from H. ducreyi ITM 5535 obtained after treatment with anhydrous hydrazine gave evidence for the presence of a sialylated major compound, Neu5Ac alpha(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Gal p- (1-->4)-D-alpha-D-Hepp-(1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp -(1-->2)-L- alpha-D-Hepp-(1-->3)]4)-L-alpha-D-Hepp-Kdo(P)-O-deacylated lipid A (Neu5Ac stands for N-acetylneuraminic acid). However, an even larger oligosaccharide could be isolated from all strains as a minor component, viz., the undecasaccharide beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-d-Galp-(1-->4)-beta-D-glcNAcp-(1-->3)-beta-D-Galp-(1-->4)-D-alpha-D-Hepp-(1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)]4-L-alpha-D-Hepp-Kdo, which represents an N-acetyl lactosamine disaccharide unit elongation of the LOS outer core. No Sialylation of this latter minor component undecasaccharide was detected.     

Measurement of beta(1-->3)-glucans in occupational and home environments with an inhibition enzyme immunoassay.

beta (1-->3)-Glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms. An inhibition enzyme immunoassay (EIA) was developed for the quantitation of beta (1-->3)-glucans in dust samples from occupational and residential environments. Immunospecific rabbit antibodies were produced by immunization with bovine serum albumin-conjugated laminarin [beta (1-->3)-glucan] and affinity chromatography on epoxy-Sepharose-coupled beta (1-->3)-glucans. The laminarin-based calibration curve in the inhibition EIA ranged from approximately 40 to 3,000 ng/ml (15 to 85% inhibition). Another beta (1-->3)-glucan (curdlan) showed a similar inhibition curve but was three to five times less reactive on a weight basis. Pustulan, presumed to be a beta (1-->6)-glucan, showed a parallel dose-response curve at concentrations 10 times higher than that of laminarin. Control experiments with NaIO4 and beta (1-->3)-glucanase treatment to destroy beta (1-->6)- and beta (1-->3)-glucan structures, respectively, indicate that the immunoreactivity of pustulan in the assay was due to beta (1-->3)-glucan and not to beta (1-->6)-glucan structures. Other polysaccharides, such as mannan and alpha (1-->6)-glucan, did not react in the inhibition EIA. Beta (1-->3)-Glucan extraction of dust samples in water (with mild detergent) was performed by heat treatment (120 degrees C) because aqueous extracts obtained at room temperature did not contain detectable beta (1-->3)-glucan levels. The assay was shown to detect heat-extractable beta (1-->3)-glucan in dust samples collected in a variety of occupational and environmental settings. On the basis of duplicate analyses of dust samples, a coefficient of variation of approximately 25% was calculated. It was concluded that the new inhibition EIA offers a useful method for indoor beta (1-->3)-glucan exposure assessment.     

The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification.

Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S. Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with [14C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change.     

Crystal structure of yeast tRNAAsp: atomic coordinates

The atomic coordinates of yeast aspartic acid transfer RNA, as determined from a crystallographic investigation to 3 A resolution, are presented. In the ribose phosphate backbone sugars are in the C(3')-endo pucker, except for residues A7, A9, D16, G17, G18, D19, C20, U48, A58, and U60 which are in the C(2')-endo pucker. A least-squares superposition of the phosphorus atoms of yeast tRNAAsp and yeast tRNAPhe enlightens both an overall structural similarity and significant conformational differences. The largest deviations occur in the D-loop and the anticodon region.     

Suppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes.

A superfamily of transport proteins, which includes the lactose permease of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E-R/K-X-G-R/K-R/K, in the loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. Previous analysis of this motif in the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995) has shown that the conserved glycine residue found at the first position in the motif (i.e., Gly-64) is important for transport function. Every substitution at this site, with the exception of alanine, greatly diminished lactose transport activity. In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function. Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64. The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 --> Ser, Leu, or Thr; Phe-29 --> Ser; Ala-50 --> Thr, Cys-154 --> Gly; Cys-234 --> Phe; Gln-241 --> Leu; Phe-261 --> Val; Thr-266 --> Iso; Val-367 --> Glu; and Ala-369 --> Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The apparent Km values of the Cys-64, Ser-64, and Val-64 parental strains were 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent Km of the wild-type permease (1.4 mM). In contrast, the Vmax values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min x mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min x mg of protein). The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains. Taken together, these results support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane. According to our tertiary model, this site is at an interface between the two halves of the protein.     

Structure and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine bilayer membranes.

The structural and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine (C(18):C(2)-PC) were studied as a function of hydration. A combination of differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the phase behavior of C(18):C(2)-PC. At low hydration (e.g., 20% H2O), the differential scanning calorimetry heating curve shows a single reversible endothermic transition at 44.6 degrees C with transition enthalpy delta H = 6.4 kcal/mol. The x-ray diffraction pattern at -8 degrees C shows a lamellar structure with a small bilayer periodicity d = 46.3 A and two wide angle reflections at 4.3 and 3.95 A, characteristic of a tilted chain, L beta' bilayer gel structure. Above the main transition temperature, a liquid crystalline L alpha phase is observed with d = 53.3 A. Electron density profiles at 20% hydration suggest that C(18):C(2)-PC forms a fully interdigitated bilayer at -8 degrees C and a noninterdigitated, liquid crystalline phase above its transition temperature (T > Tm). Between 30 and 50% hydration, on heating C(18):C(2)-PC converts from a highly ordered, fully interdigitated gel phase (L beta') to a less ordered, interdigitated gel phase (L beta), which on further heating converts to a noninterdigitated liquid crystalline L alpha phase. However, the fully hydrated (> 60% H2O) C(18):C(2)-PC, after incubation at 0 degrees C, displays three endothermic transitions at 8.9 degrees C (transition I, delta H = 1.6 kcal/mol), 18.0 degrees C (transition II), and 20.1 degrees C (transition III, delta HII+III = 4.8 kcal/mol). X-ray diffraction at -8 degrees C again showed a lamellar gel phase (L beta') with a small periodicity d = 52.3 A. At 14 degrees C a less ordered, lamellar gel phase (L beta) is observed with d = 60.5 A. However, above the transition III, a broad, diffuse reflection is observed at approximately 39 A, consistent with the presence of a micellar phase. The following scheme is proposed for structural changes of fully hydrated C(18):C(2)-PC, occurring with temperature: L beta' (interdigitated)-->L beta (interdigitated)-->L alpha(noninterdigitated)-->Micelles. Thus, at low temperature C(18):C(2)-PC forms a bilayer gel phase (L beta') at all hydrations, whereas above the main transition temperature it forms a bilayer liquid crystalline phase L alpha at low hydrations and a micellar phase at high hydrations (> 60 wt% water).     

Structure of ten free N-glycans in ripening tomato fruit. Arabinose is a constituent of a plant N-glycan.

The concentration-dependent stimulatory and inhibitory effect of N-glycans on tomato (Lycopersicon esculentum Mill.) fruit ripening was recently reported (B. Priem and K.C. Gross [1992] Plant Physiol 98: 399-401). We report here the structure of 10 free N-glycans in mature green tomatoes. N-Glycans were purified from fruit pericarp by ethanolic extraction, desalting, concanavalin A-Sepharose chromatography, and amine-bonded silica high performance liquid chromatography. N-Glycan structures were determined using 500 MHz 1H-nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and glycosyl linkage methylation analysis by gas chromatography-mass spectrometry. A novel arabinosyl-containing N-glycan, Man alpha 1-->6(Ara alpha 1-->2)Man beta 1-->4GlcNAc beta 1-->4(Fuc alpha 1-->3)GlcNAc, was purified from a retarded concanavalin A fraction. The location of the arabinosyl residue was the same as the xylosyl residue in complex N-glycans. GlcNAc[5']Man3(Xyl)GlcNAc(Fuc)GlcNAc and GlcNAc[5']Man2GlcNAc(Fuc)GlcNAc were also purified from the weakly retained fraction. The oligomannosyl N-glycans Man5GlcNAc, Man6GlcNAc, Man7GlcNAc, and Man8GlcNAc were purified from a strongly retained concanavalin A fraction. The finding of free Man5GlcNAc in situ was important physiologically because previously we had described it as a promoter of tomato ripening when added exogenously. Mature green pericarp tissue contained more than 1 microgram of total free N-glycan/g fresh weight. Changes in N-glycan composition were determined during ripening by comparing glycosyl and glycosyl-linkage composition of oligosaccharidic extracts from fruit at different developmental stages. N-Glycans were present in pericarp tissue at all stages of development. However, the amount increased during ripening, as did the relative amount of xylosyl-containing N-glycans.     

Spectrally silent transitions in the bacteriorhodopsin photocycle.

The photocycle kinetics of bacteriorhodopsin were analyzed from 0 to 40 degrees C at 101 wavelengths (330-730 nm). The data can be satisfactorily approximated by eight exponents. The slowest component (half-time 20 ms at 20 degrees C) belongs to the 13-cis cycle. The residual seven exponentials that are sufficient to describe the all-trans photocycle indicate that at least seven intermediates of the all-trans cycle must exist, although only five spectrally distinct species (K, L, M, N, and O) have been identified. These seven exponentials and their spectra at different temperatures provide the basis for the discussion of various kinetic schemes of the relaxation. The simplest model of irreversible sequential transitions includes after the first K--> L step the quasiequilibria of L<-->M, M<-->N, and N<-->O intermediates. These quasiequilibria are controlled by rate-limiting dynamics of the protein and/or proton transfer steps outside the chromophore region. Thus there exists an apparent kinetic paradox (i.e., why is the number of exponents of relaxation (at least seven) higher than the number of distinct spectral intermediates (only five)), which can be explained by assuming that some of the transitions correspond to changes in the quasiequilibria between spectrally distinct intermediates (i.e., are spectrally silent).     

Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians.

The autosomal recessive trait of thiopurine S-methytransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine. To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G460-->A and A719-->G) producing amino acid changes at codons 154 (Ala-->Thr) and 240 (Tyr--> Cys), differing from the rare mutant TPMT allele we previously identified (i.e., TPMT*2 with only G238-->C). Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G460-->A, ninefold reduction; A719-->G, 1.4-fold reduction), while the presence of both mutations leads to complete loss of activity. Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians.     

The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ.     

Electrogenicity of the sodium transport pathway in the Na,K-ATPase probed by charge-pulse experiments.

A charge-pulse technique was designed to measure charge movements in the Na-transport mode of the Na,K-ATPase in membrane fragments adsorbed to a planar lipid bilayer with high time resolution. 1) Na+ transport was measured as a function of membrane potential, and 2) voltage-dependent extracellular ion binding and release were analyzed as a function of Na+ concentration and membrane potential. The results could be fitted and explained on the basis of a Post-Albers cycle by simulations with a mathematical model. The minimal reaction sequence explaining the electrogenicity of the pump consists of the following steps: (Na3)E1-P <--> P-E2(Na3) <--> P-E2(Na2) <--> P-E2(Na) <--> P-E2. The conformational change, E1 to E2, is electrogenic (beta 0 < or = 0.1) and the rate-limiting step of forward Na+ transport with a rate constant of 25 s-1 (T = 20 degrees C). The first ion release step, P-E2(Na3) <--> P-E2(Na2), is the major charge translocating process (delta 0 = 0.65). It is probably accompanied by a protein relaxation in which the access structure between aqueous phase and binding site reduces the dielectric distance. The release of the subsequent Na+ ions has a significantly lower dielectric coefficient (delta1 = delta 2 = 0.2). Compared with other partial reactions, the ion release rates are fast (1400 s-1, 700 s-1, and 4000 s-1). On the basis of these findings, a refined electrostatic model of the transport cycle is proposed.     

Glucocerebrosidase mutations in Gaucher disease.

BACKGROUND: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. MATERIALS AND METHODS: The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. RESULTS: Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C-->A transversion at cDNA nt 644 (Ala176-->Asp), a C-->A transversion at cDNA nt 661 that resulted in a (Pro182-->Thr), and a G-->A transition at cDNA nt 721 (Gly202-->Arg). Two missense mutations were found in exon 7: a G-->A transition at cDNA nt 887 (Arg257-->Gln) and a C-->T at cDNA nt 970 (Arg285-->Cys). Two missense mutations were found in exon 9: a T-->G at cDNA nt 1249 (Trp378-->Gly) and a G-->A at cDNA nt 1255 (Asp380-->Asn). In addition to these disease-producing mutations, a silent C-->G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. CONCLUSIONS: The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.     

Mutagenesis supports water mediated recognition in the trp repressor-operator system.

High resolution crystallographic analysis of the trp repressor-operator complex indicates that the principal determinants of specificity are water mediated hydrogen bonds between the helix-turn-helix and the identity elements of the operator. One such hydration site involves a conserved G-C base pair (designated G6) six nucleotides away from the dyad which, if changed symmetrically to any other pair (e.g. G6-->A) reduces affinity to nonspecific levels. This same water site also contacts the conserved A5 which, if changed to G (mutation A5-->G), also diminishes affinity. The stereochemistry of the water mediated hydrogen bonding system predicts that the severe deterioration of in vitro binding caused by G6-->A should be reverted by a second deleterious mutation A5-->G. This proved to be the case. No other second mutation at conserved operator position 5 or 7 (flanking the G6-->A) reversed the effect of G6-->A.     

Weakening of the interface between adjacent catalytic chains promotes domain closure in Escherichia coli aspartate transcarbamoylase.

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp-100, from one catalytic chain, is involved in stabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain. Replacement of Asp-100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158). In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1-C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp-100-->Ala mutation was introduced into the Glu-50-->Ala holoenzyme, a mutant in which domain closure is impaired. The Glu-50/Asp-100-->Ala enzyme is fourfold more active than the Glu-50-->Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp-100-->Ala mutation restores the ability of the Glu-50-->Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA). At subsaturating concentrations of aspartate, the Glu-50/Asp-100-->Ala enzyme is activated more by ATP than the Glu-50-->Ala enzyme and is also inhibited more by CTP than either the wild-type or the Glu-50-->Ala enzyme. As opposed to the wild-type enzyme, the Glu-50/Asp-100-->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu-50/Asp-100-->Ala enzyme by solution X-ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild-type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA. However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu-50/Asp-100-->Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu-50-->Ala and wild-type enzymes. Solution X-ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu-50/Asp-100-->Ala enzyme.     

The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site.

The beta-subunit associated with the catalytic (alpha) subunit of the mammalian Na+, K(+) -ATPase is a transmembrane glycoprotein with three extracellularly located N-glycosylation sites. Although beta appears to be essential for a functional enzyme, the role of beta and its sugars remains unknown. In these studies, steady-state and dynamic fluorescence measurements of the fluorophore lucifer yellow (LY) covalently linked to the carbohydrate chains of beta have demonstrated that the bound probes are highly solvent exposed but restricted in their diffusional motions. Furthermore, the probes' environments on beta were not altered by Na+ or K+ or ouabain-induced enzyme conformational changes, but both divalent cation and oligomycin addition evoked modest changes in LY fluorescence. Frequency domain measurements reflecting the Förster fluorescence energy transfer (FET) occurring between anthroylouabain (AO) bound to the cardiac glycoside receptor site on alpha and the carbohydrate-linked LY demonstrated their close proximity (18 A). Additional FET determinations made between LY as donor and erythrosin-5-isothiocyanate, covalently bound at the enzyme's putative ATP binding site domain, indicated that a distance of about 85 A separates these two regions and that this distance is reduced upon divalent cation binding and increased upon the Na+E1-->K+E2 conformational transition. These data suggest a model for the localization of the terminal moieties of the oligosaccharides that places them, on average, about 18 A from the AO binding site and this distance or less from the extracellular membrane surface.