Proton nmr relaxation study of the dynamics of anthopleurin-A in solution

Spin-spin and spin-lattice 1H-nmr relaxation times of the sea anemone polypeptide anthopleurin-A were measured at frequencies of 200, 300, 400, and 500 MHz. Relaxation times were fitted iteratively by least squares regression to the isotropic tumbling model, Woessner's model for anisotropic motion, and Lipari and Szabo's "model-independent" model. Data for aromatic and aliphatic methine protons could not be fitted satisfactority using the isotropic model. Good fits were obtained, however, using the model-independent approach, indicating that high-frequency internal motions of the polypeptide backbone were significant. In addition, a range of tau c values from 2.2 to 3.2 ns was obtained for various methine protons, suggesting that overall rotational reorientation of the molecule was anisotropic. Methyl group relaxation data were fitted satisfactorily by Woessner's model. Some assessment has been made of the effect of experimental errors on the quality of fit to the data, as well as of the contribution of experimental values at certain frequencies to definition of the spectral density function.     

Backbone folding of the polypeptide cardiac stimulant anthopleurin-A determined by nuclear magnetic resonance, distance geometry and molecular dynamics

The solution conformation of the cardiac stimulatory sea anemone polypeptide anthopleurin-A has been characterised using distance geometry and restrained molecular dynamics calculations. A set of 253 approximate interproton distance restraints and 14 peptide backbone torsion angle restraints derived from two-dimensional 1H-NMR spectra at 500 MHz were used as input for these calculations. 13 structures generated by either metric matrix or variable target function distance geometry calculations were refined using energy minimisation and restrained molecular dynamics. The resulting structures contain a region of twisted antiparellel beta-sheet to which two separate regions of unordered chain are linked by three disulphide bonds. Two loops, one including Pro-41 and the other encompassing residues 10-18, are poorly defined by the NOE data.     

Amino acid similarity matrices based on force fields

MOTIVATION: We propose a general method for deriving amino acid substitution matrices from low resolution force fields. Unlike current popular methods, the approach does not rely on evolutionary arguments or alignment of sequences or structures. Instead, residues are computationally mutated and their contribution to the total energy/score is collected. The average of these values over each position within a set of proteins results in a substitution matrix. RESULTS: Example substitution matrices have been calculated from force fields based on different philosophies and their performance compared with conventional substitution matrices. Although this can produce useful substitution matrices, the methodology highlights the virtues, deficiencies and biases of the source force fields. It also allows a rather direct comparison of sequence alignment methods with the score functions underlying protein sequence to structure threading. AVAILABILITY: Example substitution matrices are available from http://www.rsc.anu.edu.au/~zsuzsa/suppl/matrices.html. SUPPLEMENTARY INFORMATION: The list of proteins used for data collection and the optimized parameters for the alignment are given as supplementary material at http://www.rsc.anu.edu.au/~zsuzsa/suppl/matrices.html.     

Protein fold recognition without Boltzmann statistics or explicit physical basis.

We present a fast method for finding optimal parameters for a low-resolution (threading) force field intended to distinguish correct from incorrect folds for a given protein sequence. In contrast to other methods, the parameterization uses information from >10(7) misfolded structures as well as a set of native sequence-structure pairs. In addition to testing the resulting force field's performance on the protein sequence threading problem, results are shown that characterize the number of parameters necessary for effective structure recognition.     

Efficacy and tolerability of potato juice in dyspeptic patients: a pilot study.

In Europe, use of potatoes (Solani tuberosi tuberecens) is a traditional remedy for stomach complaints. We performed a pilot study on the effectiveness and tolerability of freshly squeezed potato juice in patients suffering from dyspeptic symptoms. After informed written consent, 44 patients with various dyspeptic symptoms were enrolled, to drink for 1 week twice daily 100ml or more of potato juice (Biotta, if complaints persisted, a further 100ml was recommended. Validated outcome measures included the gastrointestinal symptom (GIS) profile, a disease-specific health assessment questionnaire (QOLRAD) and self-rated treatment success (efficacy, a 5-point Likert Scale). The study was completed by 42 patients. The GIS and QOLRAD scores improved significantly by 41.9+/-40.8% (p=0.001) and 50.7+/-36.1% (p<0.001), respectively (ITT); the absolute values changed from 11.5+/-5.1 to 6.3+/-5.3 (GIS) and 62.0+/-25.7 to 32.0+/-28.8 (QORAD). Sixteen, 18 and 26 patients, respectively, rated the effectiveness of the treatment as very good or good on the GIS, QOLRAD (improvements >60%) and on efficacy, respectively. Poor treatment success was achieved in 13 (GIS), 11 (QOLRAD) and 10 (Efficacy), not necessarily by the same patients. Since about two-thirds of the patients benefited to some extent from the treatment, potato juice in its present formulation may be useful for self-treatment. However, placebo-controlled studies with a longer treatment period are needed to confirm this.     

A Rapid Genetic Assay for the Identification of the Most Common Pocillopora damicornis Genetic Lineages on the Great Barrier Reef

Pocillopora damicornis (Linnaeus, 1758; Scleractinia, Pocilloporidae) has recently been found to comprise at least five distinct genetic lineages in Eastern Australia, some of which likely represent cryptic species. Due to similar and plastic gross morphology of these lineages, field identification is often difficult. Here we present a quick, cost effective genetic assay as well as three novel microsatellite markers that distinguish the two most common lineages found on the Great Barrier Reef. The assay is based on PCR amplification of two regions within the mitochondrial putative control region, which show consistent and easily identifiable fragment size differences for the two genetic lineages after Alu1 restriction enzyme digestion of the amplicons.     

The Extracellular Heme-binding Protein HbpS from the Soil Bacterium Streptomyces reticuli Is an Aquo-cobalamin Binder

The extracellular protein HbpS from Streptomyces reticuli interacts with iron ions and heme. It also acts in concert with the two-component sensing system SenS-SenR in response to oxidative stress. Sequence comparisons suggested that the protein may bind a cobalamin. UV-visible spectroscopy confirmed binding (K_d = 34 μm) to aquo-cobalamin (H₂OCbl⁺) but not to other cobalamins. Competition experiments with the H₂OCbl⁺-coordinating ligand CN⁻ and comparison of mutants identified a histidine residue (His-156) that coordinates the cobalt ion of H₂OCbl⁺ and substitutes for water. HbpS·Cobalamin lacks the Asp-X-His-X-X-Gly motif seen in some cobalamin binding enzymes. Preliminary tests showed that a related HbpS protein from a different species also binds H₂OCbl⁺. Furthermore, analyses of HbpS-heme binding kinetics are consistent with the role of HbpS as a heme-sensor and suggested a role in heme transport. Given the high occurrence of HbpS-like sequences among Gram-positive and Gram-negative bacteria, our findings suggest a great functional versatility among these proteins.     

The use of larvae or recruits in coral restoration initiatives: Symbiodinium acquisition does not differ between coral life stages in the wild

Active restoration initiatives are increasingly considered in natural resource management. Laboratory‐reared coral larvae and recruits have been proposed for stock production but it is unknown if their use impacts subsequent symbiosis once transplanted to the reef. We exposed laboratory and field settled aposymbiotic recruits (recently settled <1 month) to Symbiodinium in the wild, then analyzed the acquired communities using ITS‐2 sequencing. There was no significant difference between treatments based on overall community and diversity metrics, or differential abundance of individual taxa. These results suggest that early acquisition is analogous and thus supports the use of either life‐stage as an option for reef restoration.     

Amide proton exchange rates in cardioactive sea anemone polypeptides

Amide hydrogen exchange rates have been measured using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz for three homologous cardioactive polypeptides, anthopleurin-A from Anthopleura xanthogrammica and Anemonia sulcata toxins I and II. There are approximately 15 slowly exchanging hydrogens in each polypeptide, but the slowest exchange rates are found in ATX II, with ATX I and AP-A having rates similar to one another. The exchange rates correlate with the thermal stability of these molecules, but not with the potency and species specificity of their biological activities. The data for AP-A are interpreted in terms of a recent structural model for this polypeptide.