The lipopolysaccharide transport system (Lpt) in Gram-negative bacteria is responsible for transporting lipopolysaccharide (LPS) from the cytoplasmic surface of the inner membrane, where it is assembled, across the inner membrane, periplasm and outer membrane, to the surface where it is then inserted in the outer leaflet of the asymmetric lipid bilayer. The Lpt system consists of seven known LPS transport proteins (LptA-G) spanning from the cytoplasm to the cell surface. We have shown that the periplasmic component, LptA is able to form a stable complex with the inner membrane anchored LptC but does not interact with the outer membrane anchored LptE. This suggests that the LptC component of the LptBFGC complex may act as a dock for LptA, allowing it to bind LPS after it has been assembled at the inner membrane. That no interaction between LptA and LptE has been observed supports the theory that LptA binds LptD in the LptDE homodimeric complex at the outer membrane.Crown
The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly, L. pealei calexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.
The enzyme 2,4'-dihydroxyacetophenone dioxygenase (or DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits each containing nonhaem iron and its sequence suggests that it belongs to the cupin family of dioxygenases. By the use of limited chymotrypsinolysis, the DAD enzyme from Alcaligenes sp. 4HAP has been crystallized in a form that diffracts synchrotron radiation to a resolution of 2.2 Å.
The enzyme l-threonine dehydrogenase catalyses the NAD + -dependent conversion of l-threonine to 2-amino-3-ketobutyrate, which is the first reaction of a two-step biochemical pathway involved in the metabolism of threonine to glycine. Here, the crystallization and preliminary crystallographic analysis of l-threonine dehydrogenase (Tk-TDH) from the hyperthermophilic organism Thermococcus kodakaraensis KOD1 is reported. This threonine dehydrogenase consists of 350 amino acids, with a molecular weight of 38 kDa, and was prepared using an Escherichia coli expression system. The purified native protein was crystallized using the hanging-drop vapour-diffusion method and crystals grew in the tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 124.5, c = 271.1 Å . Diffraction data were collected to 2.6 Å resolution and preliminary analysis indicates that there are four molecules in the asymmetric unit of the crystal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.