Structure of Ala24/Asp61 --> Asp24/Asn61 substituted subunit c of Escherichia coli ATP synthase: implications for the mechanism of proton transport and rotary movement in the F0 complex

The structure of the A24D/D61N substituted subunit c of Escherichia coli ATP synthase, in which the essential carboxylate has been switched from residue 61 of the second transmembrane helix (TMH) to residue 24 of the first TMH, has been determined by heteronuclear multidimensional NMR in a monophasic chloroform/methanol/water (4:4:1) solvent mixture. As in the case of the wild-type protein, A24D/D61N substituted subunit c forms a hairpin of two extended alpha-helices (residues 5-39 and 46-78), with residues 40-45 forming a connecting loop at the center of the protein. The structure was determined at pH 5, where Asp24 is fully protonated. The relative orientation of the two extended helices in the A24D/D61N structure is different from that in the protonated form of the wild-type protein, also determined at pH 5. The C-terminal helix is rotated by 150 degrees relative to the wild-type structure, and the N-terminal helix is rotated such that the essential Asp24 carboxyl group packs on the same side of the molecule as Asp61 in the wild-type protein. The changes in helix-helix orientation lead to a structure that is quite similar to that of the deprotonated form of wild-type subunit c, determined at pH 8. When a decameric ring of c subunits was modeled from the new structure, the Asp24 carboxyl group was found to pack in a cavity at the interface between two subunits that is similar to the cavity in which Asp61 of the wild-type protein is predicted to pack. The interacting faces of the packed subunits in this model are also similar to those in the wild-type model. The results provide further evidence that subunit c is likely to fold in at least two conformational states differing most notably in the orientation of the C-terminal helix. Based upon the structure, a mechanistic model is discussed that indicates how the wild-type and A24D/D61N subunits could utilize similar helical movements during H(+) transport-coupled rotation of the decameric c ring.     

Vector competence of Coquillettidia linealis (Skuse) (Diptera: Culicidae) for Ross River and Barmah Forest viruses

Abstract Coquillettidia linealis is a severe pest on some of the Moreton Bay islands in Queensland, Australia, but little is known of its breeding habitats and biology. Because of its high abundance and its association with Ross River (RR) and Barmah Forest (BF) viruses by field isolation, its vector competence was evaluated in the laboratory by feeding dilutions of both viruses in blood. For RR, Cq. linealis was of comparable efficiency to Ochlerotatus vigilax (Skuse), recognised as being a major vector. Results were as follows for Cq. linealis and Oc. vigilax , respectively: dose to infect 50%, 10^2.2 and <10^1.7 CCID₅₀/mosquito; 88% and 90% disseminated infection at 4 days postinfection; transmission at 4 days with rates of 68−92% and 25−60%. For BF dose to infect 50%, 10^2.7 and 10^2.0; disseminated infection rates on first transmission day (day 6), 40% and 70%; transmission rates of 8−16% and 0−10%. As a capillary-tube method was used rather than suckling mice to demonstrate transmission, transmission rates may be underestimates. This, the first study of the vector competence of Cq. linealis in Australia, demonstrates that this species deserves control on the southern Moreton Bay islands.     

Inorganic Phosphate Compartmentation in the Normal Isolated Canine Brain

Abstract: In vivo ³¹P magnetic resonance spectra of 16 isolated dog brains were studied by using a 9.4-T wide-bore superconducting magnet. The observed P_i peak had an irregular shape, which implied that it represented more than one single homogeneous pool of P_i. To evaluate our ability to discriminate between single and multiple peaks and determine peak areas, we designed studies of simulated ³¹P_i spectra with the signal-to-noise (S/N) ratios ranging from ∞ to 4.4 with reference to the simulated P_i peak. For the analysis we used computer programs with a linear prediction algorithm (NMR-Fit) and a Marquardt–Levenberg nonlinear curve-fit algorithm (Peak-Fit). When the simulated data had very high S/N levels, both methods located the peak centers precisely; however, the Marquardt-Levenberg algorithm (M-L algorithm) was the more reliable at low S/N levels. The linear prediction method was poor at determining peak areas; at comparable S/N levels, the M-L algorithm determined all peak areas relatively accurately. Application of the M-L algorithm to the individual experimental in vivo dog brain data resolved the P_i peak into seven or more separate components. A composite spectrum obtained by averaging all spectral data from six of the brains with normal O₂ utilization was fitted using the M-L algorithm. The results suggested that there were eight significant peaks with the following chemical shifts: 4.07, 4.29, 4.45, 4.62, 4.75, 4.84, 4.99, and 5.17 parts per million (ppm). Although linear prediction demonstrated the presence of only three peaks, all corresponded to values obtained using the M-L algorithm. The peak indicating a compartment at 5.17 ppm (pH 7.34) was assigned to venous pH on the basis of direct simultaneous electrode-based measurements. On the basis of earlier electrode studies of brain compartmental pH, the peaks at 4.99 ppm (pH 7.16) and 4.84 ppm (pH 7.04) were thought to represent interstitial fluid and the astrocyte cytoplasm, respectively.     

1H, 13C, and 15N resonance assignments for a ferrocytochrome c553 heme by multinuclear NMR spectroscopy

A novel strategy has been used to assign the 1H, 13C, and 15N resonances of the heme in Anabaena 7120 ferrocytochrome c553. 13C[13C] double-quantum coherence spectroscopy was used to delineate the heme carbons, 1H[13C] single-bond correlation spectroscopy was used to define the attached protons, and 1H[15N] multiple-bond correlation spectroscopy was used to assign the nitrogens. 1H[13C] multiple-bond correlation spectroscopy confirmed many of the assignments. Proteins were labeled uniformly with 13C or 15N to obtain the required spectral sensitivity.     

Assignment of the 13C-NMR spectra of virgin and reactive-site modified turkey ovomucoid third domain

The virgin (reactive-site Leu18-Glu19 peptide bond intact) and modified (reactive-site Leu18-Glu19 peptide bond hydrolyzed) forms of turkey ovomucoid third domain (OMTKY3 and OMTKY3*, respectively) have been analyzed by proton-detected 1H(13C) two-dimensional single-bond correlation (1H[13C]SBC) spectroscopy. Previous 1H-nmr assignments of these proteins [A.D. Robertson, W.M. Westler, and J.L Markley (1988) Biochemistry, 27, 2519-2529; G. I. Rhyu and J. L. Markley (1988) Biochemistry, 27, 2529-2539] have been extended to directly bonded 13C atoms. Assignments have been made to 52 of the 56 backbone 13C alpha-1H units and numerous side-chain 13C-1H groups in both OMTKY3 and OMTKY3*. The largest changes in the 13C chemical shift upon conversion of OMTKY3 to OMTKY3* occur at or near the reactive site, and tend toward values observed in small peptides. Moreover, the side-chain prochiral methylene protons attached to the C gamma of Glu19 and C delta of Arg21 show nonequivalent chemical shifts in OMTKY3 but more equivalent chemical shifts in OMTKY3*. These results suggest that the reactive site region becomes less ordered upon hydrolysis of the Leu18-Glu19 peptide bond. Comparison of 13C alpha chemical shifts of OMTKY3 and bovine pancreatic trypsin inhibitor [D. Brühuiler and G. Wagner (1986) Biochemistry 25, 5839-5843; N. R. Nirmala and G. Wagner (1988) Journal of the American Chemical Society, 110, 7557-7558] with small peptide values [R. Richarz and K. Wüthrich (1978) Biopolymers, 17, 2133-2141] suggests that 13C alpha chemical shifts of residues residing in helices are generally about 2 ppm downfield of resonances from nonhelical residues.     

Studies on 3-epimeric l-2-hexulose phenylosazones. Structure and anomeric configuration of the 3,6-anhydro-osazone derivatives obtained from l-xylo- and l-lyxo-2-hexulose phenylosazone

Dehydration of the 3-epimeric 2-hexulose phenylosazones l-xylo-hexulose phenylosazone and l-lyxo-hexulose phenylosazone afforded 3,6-anhydro-l-lyxo-2-hexulose phenylosazone (2) as the preponderant isomer from both. The identity of 2 was obtained by t.l.c., and by acetylation followed by comparison of the products. Acetylation of 2 with acetic anhydride-pyridine afforded the di-O-acetyl derivative 4, and further acetylation gave the N-acetyldi-O-acetyl derivative 5. Refluxing of 2 with copper sulfate afforded a C-nucleoside analog, namely, 2-phenyl-4-α-l-threofuranosyl-1,2,3-osotriazole (6). The anomeric configuration was determined by n.m.r. spectroscopy. The stereochemical course of the dehydration process and the mass spectra of compounds 2, 4, 5, and 6 are discussed.     

Carbon-13 NMR method for the detection of correlated hydrogen exchange at adjacent backbone peptide amides and its application to hydrogen exchange in five antiparallel beta strands within the hydrophobic core of Streptomyces subtilisin inhibitor (SSI)

A novel method for monitoring proton-deuteron (H/D) exchange at backbone amides is based on the observation of H/D isotope effects on the (13)C NMR signals from peptide carbonyls. The line shape of the carbonyl (13)C(i) signal is influenced by differential H/D occupancy at the two adjacent amides: the H(N)(i)(+1) (beta site) and the H(N)(i) (gamma site). At a carbon frequency of 75.4 MHz, the H --> D isotope shifts on the (13)C signal are about 5-7 Hz for exchange at the beta site and 2 Hz or less for exchange at the gamma site. Because the effects at the two sites are additive, the time dependence of the line shape of a particular carbonyl resonance can report not only the exchange rates at the individual sites but also the level of dual exchange. Therefore, the data can be analyzed to determine the rate (k(c)) and degree of correlated exchange (X(betagamma)) at the two sites. We have applied this approach to the investigation of the pH dependence of hydrogen exchange at several adjacent residues in Streptomyces subtilisin inhibitor (SSI). Two selectively labeled SSI proteins were produced: one with selective (13)C' labeling at all valyl residues and one with selective (13)C' labeling at all leucyl residues. This permitted the direct observation by one-dimensional (13)C NMR of selected carbonyl signals from residues with slowly exchanging amides at the i and i + 1 positions. The residues investigated were located in an alpha helix and in a five-stranded antiparallel beta sheet. Samples of the two labeled proteins were prepared at various pH values, and (13)C NMR spectra were collected at 50 degrees C prior to and at various times after transferring the sample from H(2)O to (2)H(2)O. Most of the slowly exchanging amides studied were intramolecular hydrogen-bond donors. In agreement with prior studies, the results indicated that the exchange rates of the amide hydrogens in proteins are governed not only by hydrogen bonding but also by other factors. For example, the amide hydrogen of Thr34 exchanges rapidly even though it is an intramolecular hydrogen-bond donor. Over nearly the whole pH range studied, the apparent rates of uncorrelated exchange (k(beta) and k(gamma)) were proportional to [OH(-)] and the apparent rates of correlated exchange at two adjacent sites (k(c)) were roughly proportional to [OH(-)](2). This enabled us to extract the pH-independent exchange rates (k(beta) degrees , k(gamma) degrees , and k(c) degrees ). In all cases in which correlated exchange could be measured, the observed sigmoidal pH dependence of X(betagamma) could be replicated roughly from the derived pH-independent rates.     

Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination

Protocols have been developed and applied for the high-throughput production of [U-15N]- or [U-13C-, U-15N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of approximately 5, an average wet cell mass yield of approximately 9.5 g L(-1), and an average yield of approximately 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15N and 13C at levels greater than 95%. 1H-15N heteronuclear single quantum correlation spectroscopy as well as 13C; 15N-edited spectroscopy showed that the purified [U-15N]- and [U-13C, U-15N]-labeled proteins were suitable for structure analysis.