Conformational contagion in a protein: structural properties of a chameleon sequence

Certain sequences, known as chameleon sequences, take both alpha- and beta-conformations in natural proteins. We demonstrate that a wild chameleon sequence fused to the C-terminal alpha-helix or beta-sheet in foreign stable proteins from hyperthermophiles forms the same conformation as the host secondary structure. However, no secondary structural formation is observed when the sequence is attached to the outside of the secondary structure. These results indicate that this sequence inherently possesses an ability to make either alpha- or beta-conformation, depending on the sequentially neighboring secondary structure if little other nonlocal interaction occurs. Thus, chameleon sequences take on a satellite state through contagion by the power of a secondary structure. We propose this "conformational contagion" as a new nonlocal determinant factor in protein structure and misfolding related to protein conformational diseases.     

Origin of functional diversity among tetrameric voltage-gated channels

The aim of the present work is to relate functional differences of voltage-gated K(+) (K(v)), hyperpolarization-activated cyclic nucleotide-gated (HCN), and cyclic nucleotide gated (CNG) channels to differences in their amino acid sequences. By means of combined bioinformatic sequence analyses and homology modelling, we suggest that: (1) CNG channels are less voltage-dependent than K(v) channels since the charge of their voltage sensor, the S4 helix, is lower than that of K(v) channels and because of the presence of a conserved proline in the S4-S5 linker, which is quite likely to uncouple S4 from S5 and S6. (2) In HCN channels, S4 features a higher net positive charge with respect to K(v) channels and an extensive network of hydrophobic residues, which is quite likely to provide a tight coupling among S4 and the neighboring helices. We suggest insights on the gating of HCN channels and the reasons why they open with membrane hyperpolarization and with a significantly longer time constant with respect to other channels.     

Modeling and analysis of molecularinteraction between Smurf1-WW2 domain and various isoforms of LIM mineralization protein

LIM Mineralization Protein-1 (LMP-1) has been cloned and shown to be osteoinductive. Our efforts to understand the mode of action of LMP-1 led to the determination that LMP-1 interacts with Smad Ubiquitin Regulatory Factor-1 (Smurf1). Smurf1 targets osteogenic Smads, Smad1/5, for ubiquitin-mediated proteasomal degradation. Smurf1 interaction with LMP-1 or Smads is based on the presence of unique WW-domain interacting motif in these target molecules. By performing site-directed mutagenesis and binding studies in vitro on purified recombinant proteins, we identified a specific motif within the osteogenic region of several LMP isoforms that is necessary for Smurf1 interaction. Similarly, we have identified that the WW2 domain of Smurf1 is necessary for target protein interaction. Here, we present a homology-based modeling of the Smurf1 WW2 domain and its interacting motif of LMP-1. We performed computational docking of the interacting domains in Smurf1 and LMPs to identify the key amino acid residues involved in their binding regions. In support of the computational predictions, we also present biochemical evidence supporting the hypothesis that the physical interaction of Smurf1 and osteoinductive forms of LMP may prevent Smurf1 from targeting osteogenic Smads by ubiquitin-mediated proteasomal degradation.     

MiniReview: bioinformatic study of bile responses in Campylobacterales

Campylobacter, Helicobacter and Wolinella are genera of the order Campylobacterales, belonging to the class Epsilonproteobacteria. Their habitats are various niches in the gastrointestinal tract of higher animals, where they may come into contact with bile. Microorganisms in these environments require mechanisms of resistance to the surface-active amphipathic molecules with potent antimicrobial activities present in bile. This review summarizes current knowledge on the molecular responses to bile by Campylobacterales and other bacterial species that inhabit the intestinal tract and belong to the phyla Proteobacteria, Bacteriodetes, Firmicutes and Actinobacteria. To date, 125 specific genes have been implicated in bile responses, of which 10 are found in Campylobacterales. Genome database searches, analyses of protein sequence and domain similarities, and gene ontology data integration were performed to compare the responses to bile of these bacteria. The results showed that 33 proteins of bacteria belonging to the four phyla had similarities equal to or greater than 50-46% proteins of Campylobacterales. Domain architecture analyses revealed that 151 Campylobacterales proteins had similar domain composition and organization to 60 proteins known to participate in the tolerance to bile in other bacteria. The proteins CmeB, CmeF and CbrR of Campylobacter jejuni involved in bile tolerance were homologous to 42 proteins identified in the Proteobacteria, Bacteriodetes and Firmicutes. On the other hand, the proteins CiaB, CmeA, CmeC, CmeD, CmeE and FlaAsigma(28) also involved in the response to bile of C. jejuni, did not have homologues in other bacteria. Among the bacteria inhabiting the gastrointestinal tract, the Campylobacterales seem to have evolved some mechanisms of bile resistance similar to those of other bacteria, as well as other mechanisms that appear to be characteristic of this order.     

<Emphasis Type="Italic">ATHB23</Emphasis>, an <Emphasis Type="Italic">Arabidopsis</Emphasis> class I homeodomain-leucine zipper gene,is expressed in the adaxial region of young leaves

Homeobox genes are essential regulators of plant development. ATHB23, a class I homeodomain leucine zipper gene of Arabidopsis, was found to be induced by treatment with the phytohormone gibberellin (GA). In order to clarify its role in development, we performed a histochemical analysis of transgenic plants containing a construct with a GUS::GFP reporter under the control of the 1.5 kb upstream region of ATHB23. The construct was mainly expressed in young leaves and the styles of flowers but not in mature leaves. Microscopic examination of young leaves revealed that it was expressed in the adaxial domain of leaf primordia and the rib meristem. Expression of ATHB23, like that of GA5 encoding GA 20-oxidase, was reduced in mutants related to adaxial-abaxial leaf polarity (phb-1d, se-2, and kan1 kan2). Reduced expression of the GUS::GFP reporter gene was also observed in an se-2 background. These results indicate that ATHB23 is under the control of GA and other activators such as PHB, and is involved in establishing polarity during leaf development.     

Behavioural syndromes in Merriam's kangaroo rats (Dipodomys merriami): a test of competing hypotheses

Behavioural syndromes, correlations of behaviours conceptually analogous to personalities, have been a topic of recent attention due to their potential to explain trade-offs in behavioural responses, apparently maladaptive behaviour and limits to plasticity. Using Merriam's kangaroo rats (Dipodomys merriami), we assessed the explanatory power and generality of hypothesized syndrome structures derived from the literature and the natural history of the species. Several aspects of functionally distinct behavioural responses of D. merriami were quantified. Syndrome structures were compared using structural equation modelling and model selection procedures. A domain-general behavioural syndrome incorporating cross-functional relationships between measures of boldness, agonistic behaviour, flexibility and food hoarding best explained the data. This pattern suggests that D. merriami behaviours should not be viewed as discrete elements but as components of a multivariate landscape. Our results support arguments that a lack of independence between behaviours may be a general aspect of behavioural phenotypes and suggest that the ability of D. merriami's behaviour to respond to selection may be constrained by underlying connections.     

Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain

beta-Barrel proteins are present in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The central component of their assembly machinery is called Omp85 in bacteria. Omp85 is predicted to consist of an integral membrane domain and an amino-terminal periplasmic extension containing five polypeptide-transport-associated (POTRA) domains. We have addressed the function of these domains by creating POTRA domain deletions in Omp85 of Neisseria meningitidis. Four POTRA domains could be deleted with only slight defects in Omp85 function. Only the most carboxy-terminal POTRA domain was essential, as was the membrane domain. Thus, similar to the mitochondrial Omp85 homologue, the functional core of bacterial Omp85 consists of its membrane domain and a single POTRA domain, that is, POTRA5.     

Fusion of carbohydrate binding modules from <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis> with a family 10 xylanase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> S7

Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)₆ tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature and pH for the activity of the xylanase did not change.     

Specificity of Phage Display Selected Peptides for Modified Anticodon Stem and Loop Domains of tRNA

Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA^Lys3 having 2-thiouridine (s²U₃₄) and pseudouridine (Ψ₃₉), bound the modified human ASL^Lys3(s²U₃₄;Ψ₃₉) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL^Lys3(s²U₃₄;Ψ₃₉), followed by the hypermodified ASL^Lys3 (mcm⁵s²U₃₄;ms²t⁶A₃₇) and the unmodified ASL^Lys3, but bound poorly to singly modified ASL^Lys3 constructs (Ψ₃₉, ms²t⁶A_37, s²U₃₄), ASL^Lys1,2 (t⁶A₃₇) and Escherichia coli ASL^Glu (s²U₃₄). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.