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MreB, MreC and MreD are essential cell shape-determining morphogenetic proteins in Gram-positive and in Gram-negative bacteria. While MreB, the bacterial homologue of the eukaryotic cytoskeletal protein actin, has been extensively studied, the roles of MreC and MreD are less well understood. They both are transmembrane proteins. MreC has a predicted single transmembrane domain and the C-terminal part outside the cell membrane. MreC probably functions as a link between the intracellular cytoskeleton and the cell wall synthesizing machinery which is located at the outer surface of the cell membrane. Also proteins involved in cell wall synthesis participate in cell morphogenesis. How these two processes are coordinated is, however, poorly understood. Bacillus subtilis (BS), a non-pathogenic Gram-positive bacterium, is widely used as a model for Gram-positive pathogens, e.g. Staphylococcus aureus (SA). Currently, the structures of MreC from BS and SA are not known. As part of our efforts to elucidate the structure-function relationships of the morphogenetic protein complexes in Gram-positive bacteria, we present the backbone and side chain resonance assignments of the extracytoplasmic domain of MreC from BS.

Homogenous acylation and carbanilation reactions of wood-based lignocellulosic materials have been investigated in ionic liquids. We have found that highly substituted lignocellulosic esters can be obtained under mild conditions (2 h, 70 degrees C) by reacting wood dissolved in ionic liquids with acetyl chloride, benzoyl chloride, and acetic anhydride in the presence of pyridine. In the absence of pyridine, extensive degradation of the wood components was found to occur. Highly substituted carbanilated lignocellulosic material was also obtained in the absence of base in ionic liquid. These chemical modifications were confirmed by infrared spectroscopy, (1)H NMR, and quantitative (31)P NMR of the resulting derivatives. The latter technique permitted the degrees of substitution to be determined, which were found to vary between 81% and 95% for acetylation, benzoylation, and carbanilation, accompanied by similarly high gains in weight percent values. Thermogravimetric measurements showed that the resulting materials exhibit different thermal stabilities from those of the starting wood, while differential scanning calorimetry showed discrete new thermal transitions for these derivatives. Scanning electron microscopy showed the complete absence of fibrous characteristics for these derivatives, but instead, a homogeneous porous, powdery appearance was apparent. A number of our reactions were also carried out in completely recycled ionic liquids, verifying their utility for potential applications beyond the laboratory bench.

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca(2+)-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca(2+)-loaded regulatory domain of recombinant cTnC(C35S) was observed. The changes in the NMR spectra of the N-domain of full-length cTnC(C35S), due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnC(A-Cys), where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnC(C35S) and cTnC(A-Cys). The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca(2+)-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN(3)), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.

Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections of brain. Binding to cell surface heparan sulfate is indispensable for the biological activity of HB-GAM. In the present paper we have studied the structure of recombinant HB-GAM using heteronuclear NMR. These studies show that HB-GAM contains two beta-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel beta-strands. In addition to this domain structure, HB-GAM contains the N- and C-terminal lysine-rich sequences that lack a detectable structure and appear to form random coils. Studies using CD and NMR spectroscopy suggest that HB-GAM undergoes a conformational change upon binding to heparin, and that the binding occurs primarily to the beta-sheet domains of the protein. Search of sequence data bases shows that the beta-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the beta-sheet structures found previously in midkine, a protein homologous with HB-GAM, also correspond to the TSR motif. We suggest that the TSR sequence motif found in various extracellular proteins defines a beta-sheet structure similar to that found in HB-GAM and midkine. In addition to the apparent structural similarity, a similarity in biological functions is suggested by the occurrence of the TSR sequence motif in a wide variety of proteins that mediate cell-to-extracellular matrix and cell-to-cell interactions, in which the TSR domain mediates specific cell surface binding.

Anew method for measurement of one-bond (13)C'-(13)C(alpha) scalar and dipolar couplings from a two-dimensional [(15)N, (1)H] correlation spectrum is presented. The experiment is based on multiple-quantum coherence, which is created between nitrogen and carbonyl carbon for simultaneous evolution of (15)N chemical shift and coupling between (13)C' and (13)C(alpha). Optional subspectral editing is provided by the spin-state-selective filters. The residual dipolar dipolar contribution to the (13)C'-(13)C(alpha) coupling can be measured from these simplified [(15)N, (1)H]-HSQC-like spectra. In this way, without explicit knowledge of carbon assignments, conformational changes of proteins dissolved in dilute liquid crystals can be probed conveniently, e.g., in structure activity relationship by NMR studies. The method is demonstrated with human cardiac troponin C. Copyright 1999 Academic Press.

The B0 gradient pulses can be replaced with long high-power pulses (RF-gradients) in z-rotation composite sandwiches. By doing this, practically all B0 gradient-based pulse sequences can be performed with instruments lacking a field gradient accessory. We have implemented RF-gradients into selective 1D and nonselective 2D TOCSY and NOESY experiments. The spectral quality obtained with the RF-gradient method was comparable to that of the B0 method for small- to medium-sized molecules. There are also some advantages in using RF-gradients instead of B0 gradients. There is practically no shift or coupling evolution during RF-gradient pulses. This may be significant in some experiments. Copyright 1998 Academic Press.