An ATP/2e-stoichiometry of 1 1/2 is thermodynamically possible for site 3 of oxidative phosphorylation

Free energy changes for ATP synthesis (delta GP) and 2e(-)-transfer across Site 3 (delta GR) were determined during oxidative phosphorylation by rat liver mitochondria. At static head, -delta GR/delta GP ranged narrowly between 1.55 and 1.59 with five different respiratory substrates. Thus, an ATP/2e- of 1 1/2 at Site 3 is thermodynamically possible with regards to overall reactants and products. Using nonequilibrium thermodynamics, phenomenological stoichiometries were close to 1 1/2 for all substrates suggesting that ATP/2e- at Site 3 is, in fact, 1 1/2. An ATP/2e- of 1 1/2 can only be possible if H+/O is 4 for cytochrome oxidase.     

Computational Design of a Protein-Based Enzyme Inhibitor

While there has been considerable progress in designing protein–protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding.     

Monitoring population‐level responses of marine mammals to human activities

We provide guidance for monitoring whether human activities affect the physiology or behavior of marine mammals and, if so, whether those effects may lead to changes in survival and reproduction at the population level. We suggest that four elements be included in designing and implementing such a monitoring program. The first is development of a theory of change: a set of mechanistic hypotheses that outline why a given activity might be expected to have one or more measurable effects on individuals and populations, and ideally the magnitude, timing, and duration of the effects. The second element, definition of biologically meaningful effect sizes, ultimately facilitates the development of a monitoring program that can detect those magnitudes of effect with the desired levels of precision. The third element, selection of response variables for monitoring, allows inference to whether observed changes in the status of individuals or populations are attributable to a given activity. Visual observations, passive acoustic and tagging instruments, and direct physical measurements all can provide data that facilitate quantitative hypothesis testing. The fourth element is specification of the temporal sequence of monitoring. These elements also can be used to inform monitoring of the responses of other taxonomic groups to human activities.     

N-terminal Gly(224)-Gly(411) domain in Listeria adhesion protein interacts with host receptor Hsp60

Background

Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated.

Methods

Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met₁–Pro₂₂₃) and N2 (Gly₂₂₄–Gly₄₁₁), and the ADH region contains C1 (Gly₄₁₂–Val₆₄₈) and C2 (Pro₆₄₉–Val₈₆₆). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain''s affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells.

Results

The N2 subdomain exhibited the greatest affinity for Hsp60 with a K _D of 9.50±2.6 nM. The K _D of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (P<0.05) higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells.

Conclusion

These data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies.     

A horizon scan of global conservation issues for 2012

Our aim in conducting annual horizon scans is to identify issues that, although currently receiving little attention, may be of increasing importance to the conservation of biological diversity in the future. The 15 issues presented here were identified by a diverse team of 22 experts in horizon scanning, and conservation science and its application. Methods for identifying and refining issues were the same as in two previous annual scans and are widely transferable to other disciplines. The issues highlight potential changes in climate, technology and human behaviour. Examples include warming of the deep sea, increased cultivation of perennial grains, burning of Arctic tundra, and the development of nuclear batteries and hydrokinetic in-stream turbines.     

Management implications of the biodiversity and socio-economic impacts of shrimp trawler by-catch in Bahía de Kino, Sonora, México

The shrimp fishery is the most economically important fishery in Mexico. The trawler-based portion of this fishery results in high rates of by-catch. This study quantifies and describes the biodiversity of by-catch associated with trawling in the Bahía de Kino region of Sonora, Mexico. Data were collected from 55 trawls, on six boats, over 14 nights, during November of 2003, 2004, 2006-2009. By-catch rates within trawl samples averaged 85.9% measured by weight. A total of 183 by-catch species were identified during the course of this study, including 97 species of bony fish from 43 families, 19 species of elasmobranchs from 12 families, 66 species of invertebrates from eight phyla, and one species of marine turtle; seven of the documented by-catch species are listed on the IUCN Red List, CITES, or the Mexican NOM-059-ECOL-2010; 35 species documented in the by-catch are also targeted by local artisanal fishers. Some of the species frequently captured as juveniles in the by-catch are economically important to small-scale fishers in the region, and are particularly sensitive to overexploitation due to their life histories. This study highlights the need for further research quantifying the impacts of high levels of by-catch upon small-scale fishing economies in the region and presents strong ecological and economic rationale for by-catch management within the shrimp fishery of the Gulf of California. Site-specific by-catch management plans should be piloted in the Bahía de Kino region to address the growing momentum in national and international fisheries policy regimes toward the reduction of by-catch in shrimp fisheries.