Identification of protein side chains near the membrane-aqueous interface: a site-directed spin labeling study of KcsA.

This study presents an approach to identifying surface residues on membrane proteins that are exposed toward the membrane-aqueous interface. The method employs a lipid Ni(II) chelate that localizes the metal ion to a region near the membrane-aqueous interface. Lateral diffusion of the lipid chelate results in Heisenberg exchange (HE) with nitroxide side chains in the protein only if direct contact occurs between the paramagnetic species during a collision. Thus, HE serves as a signature for residues facing the bilayer in the neighborhood of the membrane-aqueous interface. To evaluate the method, 13 surface residues on the extracellular half of KcsA, a prokaryotic potassium channel of known structure, were examined for HE with the Ni(II) chelate. The HE rate between the two species is found to depend strongly on the vertical position of the nitroxide with respect to the membrane-aqueous interface. Nitroxides introduced near the interface experience relatively high HE rates, whereas nitroxides that are immersed in the bilayer interior or sterically sheltered from collision experience low or undetectable rates. The results indicate that residues near the interface can be identified on the basis of their high rates of collision with the headgroup region of the bilayer.     

What Can We Learn from the Evolution of Protein-Ligand Interactions to Aid the Design of New Therapeutics?

Efforts to increase affinity in the design of new therapeutic molecules have tended to lead to greater lipophilicity, a factor that is generally agreed to be contributing to the low success rate of new drug candidates. Our aim is to provide a structural perspective to the study of lipophilic efficiency and to compare molecular interactions created over evolutionary time with those designed by humans. We show that natural complexes typically engage in more polar contacts than synthetic molecules bound to proteins. The synthetic molecules also have a higher proportion of unmatched heteroatoms at the interface than the natural sets. These observations suggest that there are lessons to be learnt from Nature, which could help us to improve the characteristics of man-made molecules. In particular, it is possible to increase the density of polar contacts without increasing lipophilicity and this is best achieved early in discovery while molecules remain relatively small.     

Inhibition of C3 Convertase Activity by Hepatitis C Virus as an Additional Lesion in the Regulation of Complement Components

We have previously reported that in vitro HCV infection of cells of hepatocyte origin attenuates complement system at multiple steps, and attenuation also occurs in chronically HCV infected liver, irrespective of the disease stage. However, none of these regulations alone completely impaired complement pathways. Modulation of the upstream proteins involved in proteolytic processing of the complement cascade prior to convertase formation is critical in promoting the function of the complement system in response to infection. Here, we examined the regulation of C2 complement expression in hepatoma cells infected in vitro with cell culture grown virus, and validated our observations using randomly selected chronically HCV infected patient liver biopsy specimens. C2 mRNA expression was significantly inhibited, and classical C3 convertase (C4b2a) decreased. In separate experiments for C3 convertase function, C3b deposition onto bacterial membrane was reduced using HCV infected patient sera as compared to uninfected control, suggesting impaired C3 convertase. Further, iC3b level, a proteolytically inactive form of C3b, was lower in HCV infected patient sera, reflecting impairment of both C3 convertase and Factor I activity. The expression level of Factor I was significantly reduced in HCV infected liver biopsy specimens, while Factor H level remained unchanged or enhanced. Together, these results suggested that inhibition of C3 convertase activity is an additional cumulative effect for attenuation of complement system adopted by HCV for weakening innate immune response.     

INVERTEBRATE‐MEDIATED NUTRIENT LOADING INCREASES GROWTH OF AN INTERTIDAL MACROALGA1

Even in nitrogen‐replete ecosystems, microhabitats exist where local‐scale nutrient limitation occurs. For example, coastal waters of the northeastern Pacific Ocean are characterized by high nitrate concentrations associated with upwelling. However, macroalgae living in high‐zone tide pools on adjacent rocky shores are isolated from this upwelled nitrate for extended periods of time, leading to nutrient limitation. When high‐intertidal pools are isolated during low tide, invertebrate‐excreted ammonium accumulates, providing a potential nitrogen source for macroalgae. I quantified the influence of mussels (Mytilus californianus Conrad) on ammonium accumulation rates in tide pools. I then evaluated the effects of ammonium loading by mussels on nitrogen assimilation and growth rates of Odonthalia floccosa (Esp.) Falkenb., a common red algal inhabitant of pools on northeastern Pacific rocky shores. Odonthalia was grown in artificial tide pool mesocosms in the presence and absence of mussels. Mesocosms were subjected to a simulated tidal cycle mimicking emersion and immersion patterns of high‐intertidal pools on the central Oregon coast. In the presence of mussels, ammonium accumulated more quickly in the mesocosms, resulting in increased rates of nitrogen assimilation into algal tissues. These increased nitrogen assimilation rates were primarily associated with higher growth rates. In mesocosms containing mussels, Odonthalia individuals added 41% more biomass than in mesocosms without mussels. This direct positive effect of mussels on macroalgal biomass represents an often overlooked interaction between macroalgae and invertebrates. In nutrient‐limited microhabitats, such as high‐intertidal pools, invertebrate‐excreted ammonium is likely an important local‐scale contributor to macroalgal productivity.