Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling

Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific ¹⁵N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A.     

First partial cranium of Togocetus from Kpogamé (Togo) and the protocetid diversity in the Togolese phosphate basin

Earliest cetaceans (whales) originated from the early Eocene of Indo-Pakistan, but the group dispersed through most of the oceans of the planet by the late middle to late Eocene. This late Eocene global distribution indicates that important dispersal events took place during the middle Eocene (Lutetian), a globally undersampled time interval that is well documented in the Togolese phosphate series. We report here the first discovery of a partial cetacean cranium from middle Eocene deposits of Togo (West Africa). A 3D model of the cranium and teeth was reconstructed in order to reveal hidden anatomical features. The dental and cranial characteristics of the Togolese specimen recall those of protocetid taxa described in Africa, Asia, and North America, but also display significant differences. In particular, we show that the new specimen shares a number of morphological features with the Togolese taxon Togocetus. Such a hypothesis is further supported by a cladistic analysis including 45 taxa and 167 morphological characters, which recovers the new specimen close to Togocetus as the first offshoot of protocetids. Phylogenetic analysis including all the protocetids remains of Kpogamé confirms the singular diversity of the Togolese phosphate basin, and enables to examine potential connections with faunas from contemporaneous localities in Africa.     

Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia.

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278-300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic alpha helix ranging from Phe280 to Tyr291, followed by a type I beta turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278-292 and YopD271-292, lacked helical structure, implicating the beta turn in helix stability. An interaction between YopD278-300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide-LcrH complex. These effects were not observed for the unstructured peptide, YopD278-292, which confirms that the alpha helical structure of the YopD amphipathic domain is a critical binding region of LcrH.     

Solution structure of a DNA octamer containing the Pribnow box via restrained molecular dynamics simulation with distance and torsion angle constraints derived from two-dimensional nuclear magnetic resonance spectral fitting.

The DNA octamer [d(GTATAATG].[(CATATTAC)], containing the prokaryotic upstream consensus recognition sequence, has been examined via proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra. All proton resonances, except those of H5' and H5" protons, were assigned. A temperature dependence study of one-dimensional nuclear magnetic resonance (NMR) spectra, rotating frame 2D NOE spectroscopy (ROESY), and T1 rho measurements revealed an exchange process that apparently is global in scope. Work at lower temperatures enabled a determination of structural constraints that could be employed in determination of a time-averaged structure. Simulations of the 2QF-COSY cross-peaks were compared with experimental data, establishing scalar coupling constant ranges of the individual sugar ring protons and hence pucker parameters for individual deoxyribose rings. The rings exhibit a dynamic equilibrium of N and S-type conformers with 80 to 100% populations of the latter. A program for iterative complete relaxation matrix analysis of 2D NOE spectral intensities, MARDIGRAS, was employed to give interproton distances for each mixing time. According to the accuracy of the distance determination, upper and lower distance bounds were chosen. The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force-field, which was employed for restrained molecular dynamics calculations. Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data. Several restrained molecular dynamics runs of 25 picoseconds were performed, utilizing 184 experimental distance constraints and 80 torsion angle constraints; three different starting structures were used: energy minimized A-DNA, B-DNA, and wrinkled D-DNA, another member of the B-DNA family. Convergence to similar structures obtained with root-mean-square deviations between resulting structures of 0.37 to 0.92 A for the central hexamer of the octamer. The average structure from the nine different molecular dynamics runs was subjected to final restrained energy minimization. The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and exhibited a substantial improvement in the 2D NOE sixth-root residual index in comparison with the starting structures. An approximation of the structure in the terminal base-pairs, which displayed experimental evidence of fraying, was made by maintaining the structure of the inner four base-pairs and performing molecular dynamics simulations with the experimental structural constraints observed for the termini.(ABSTRACT TRUNCATED AT 400 WORDS)     

The "two-state folder" MerP forms partially unfolded structures that show temperature dependent hydrogen exchange

We have analysed the folding energy landscape of the 72 amino acid protein MerP by monitoring native state hydrogen exchange as a function of temperature in the range of 7-55 degrees C. The temperature dependence of the hydrogen exchange has allowed us to determine DeltaG, DeltaH and DeltaC(p) values for the conformational processes that permit hydrogen exchange. When studied with the traditional probes, fluorescence and CD, MerP appears to behave as a typical two-state protein, but the results from the hydrogen exchange analysis reveal a much more complex energy landscape. Analysis at the individual amino acid level show that exchange is allowed from an ensemble of partially unfolded structures (i.e. intermediates) in which the stabilities at the amino acid level form a broad distribution throughout the protein. The formation of partially unfolded structures might contribute to the unusually slow folding of MerP.     

NMR studies of calcium-binding to mutant alpha-spectrin EF-hands

The co-operative calcium binding mechanism of the two C-terminal EF-hands of human alphaII-spectrin has been investigated by site-specific mutagenesis and multi-dimensional NMR spectroscopy. To analyse the calcium binding of each EF-hand independently, two mutant structures (E33A and D69S) of wild type alpha-spectrin were prepared. According to NMR analysis both E33A and D69S were properly folded. The unmutated EF-hand in these mutants remained nearly intact and active in calcium binding, whereas the mutated EF-hand lost its affinity for calcium completely. The apparent calcium binding affinity of the E33A mutant was much lower compared to the D39S mutant (approximately 2470 microM and approximately 240 microM, respectively). When the chemical shift perturbations were followed upon calcium titration, a positive correlation between the D69S mutant and the binding of the first calcium ion to the wild type was revealed. These observations showed that the first EF-hand in spectrin binds the first calcium ion and thereby triggers a conformational change that allows the second calcium ion to bind to the other EF-hand.     

New data on the Dyrosauridae (Crocodylomorpha) from the Paleocene of Togo

Following our fieldwork in Paleogene deposits of Togo, we herein report cranial as well as postcranial elements belonging to the family Dyrosauridae. This assemblage is dated to the Late Paleocene (Thanetian) from two quarries in southern Togo. The specimens include a partial skull presenting two large supratemporal fossae and a massive occipital condyle; long and slender isolated teeth; amphicoelous vertebrae including several articulated ones; and two osteoderms devoid of carina. The morphology of the partial skull reveals similarities with some African longirostrine forms such as Rhabdognathus spp., although this attribution cannot be confirmed. Longirostrine forms, known in the late Paleocene and early Eocene of the Iullemmeden basin (Nigeria, Niger, Mali, Algeria) and in the phosphates of Morocco and Tunisia, is only represented in Thanetian levels in Togo. Different palaeoenvironmental settings seem to have characterized the various African basins during the lower Eocene, with consequences for the geographic distribution of dyrosaurids. These dyrosaurid remains confirm the presence of the family in Togo during the Paleocene and underline the fossiliferous potential of the coastal sedimentary basin in Togo and in the bay of Benin.     

GuHCl and NaCl-dependent hydrogen exchange in MerP reveals a well-defined core with an unusual exchange pattern

We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (DeltaG(HX)) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions.     

Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus

Wood cells, unlike most other cells in plants, grow by a unique combination of intrusive and symplastic growth. Fibers grow in diameter by diffuse symplastic growth, but they elongate solely by intrusive apical growth penetrating the pectin-rich middle lamella that cements neighboring cells together. In contrast, vessel elements grow in diameter by a combination of intrusive and symplastic growth. We demonstrate that an abundant pectin methyl esterase (PME; EC 3.1.1.11) from wood-forming tissues of hybrid aspen (Populus tremula x tremuloides) acts as a negative regulator of both symplastic and intrusive growth of developing wood cells. When PttPME1 expression was up- and down-regulated in transgenic aspen trees, the PME activity in wood-forming tissues was correspondingly altered. PME removes methyl ester groups from homogalacturonan (HG) and transgenic trees had modified HG methylesterification patterns, as demonstrated by two-dimensional nuclear magnetic resonance and immunostaining using PAM1 and LM7 antibodies. In situ distributions of PAM1 and LM7 epitopes revealed changes in pectin methylesterification in transgenic trees that were specifically localized in expanding wood cells. The results show that en block deesterification of HG by PttPME1 inhibits both symplastic growth and intrusive growth. PttPME1 is therefore involved in mechanisms determining fiber width and length in the wood of aspen trees.