Cellobiose Dehydrogenase Aryl Diazonium Modified Single Walled Carbon Nanotubes: Enhanced Direct Electron Transfer through a Positively Charged Surface

One of the challenges in the field of biosensors and biofuel cells is to establish a highly efficient electron transfer rate between the active site of redox enzymes and electrodes to fully access the catalytic potential of the biocatalyst and achieve high current densities. We report on very efficient direct electron transfer (DET) between cellobiose dehydrogenase (CDH) from Phanerochaete sordida (PsCDH) and surface modified single walled carbon nanotubes (SWCNT). Sonicated SWCNTs were adsorbed on the top of glassy carbon electrodes and modified with aryl diazonium salts generated in situ from p-aminobenzoic acid and p-phenylenediamine, thus featuring at acidic pH (3.5 and 4.5) negative or positive surface charges. After adsorption of PsCDH, both electrode types showed excellent long-term stability and very efficient DET. The modified electrode presenting p-aminophenyl groups produced a DET current density of 500,mu A cm(-2) at 200 mV vs normal hydrogen reference electrode (NHE) in a 5 mM lactose solution buffered at pH 3.5. This is the highest reported DET value so far using a CDH modified electrode and comes close to electrodes using mediated electron transfer. Moreover, the onset of the electrocatalytic current for lactose oxidation started at 70 mV vs NHE, a potential which is 50 mV lower compared to when unmodified SWCNTs were used. This effect potentially reduces the interference by oxidizable matrix components in biosensors and increases the open circuit potential in biofuel cells. The stability of the electrode was greatly increased compared with unmodified but cross-linked SWCNTs electrodes and lost only 15% of the initial current after 50 h of constant potential scanning

E9-Im9 Colicin DNase−Immunity Protein Biomolecular Association in Water: A Multiple-Copy and Accelerated Molecular Dynamics Simulation Study

Protein−protein transient and dynamic interactions underlie all biological processes. The molecular dynamics (MD) of the E9 colicin DNase protein, its Im9 inhibitor protein, and their E9-Im9 recognition complex are investigated by combining multiple-copy (MC) MD and accelerated MD (aMD) explicit-solvent simulation approaches, after validation with crystalline-phase and solution experiments. Im9 shows higher flexibility than its E9 counterpart. Im9 displays a significant reduction of backbone flexibility and a remarkable increase in motional correlation upon E9 association. Im9 loops 23−31 and 54−64 open with respect to the E9-Im9 X-ray structure and show high conformational diversity. Upon association a large fraction (∼20 nm2) of E9 and Im9 protein surfaces become inaccessible to water. Numerous salt bridges transiently occurring throughout our six 50 ns long MC-MD simulations are not present in the X-ray model. Among these Im9 Glu31−E9 Arg96 and Im9 Glu41−Lys89 involve interface interactions. Through the use of 10 ns of Im9 aMD simulation, we reconcile the largest thermodynamic impact measured for Asp51Ala mutation with Im9 structure and dynamics. Lys57 acts as an essential molecular switch to shift Im9 surface loop towards an ideal configuration for E9 inhibition. This is achieved by switching Asp60−Lys57 and Asp62−Lys57 hydrogen bonds to Asp51−Lys57 salt bridge. E9-Im9 recognition involves shifts of conformational distributions, reorganization of intramolecular hydrogen bond patterns, and formation of new inter- and intramolecular interactions. The description of key transient biological interactions can be significantly enriched by the dynamic and atomic-level information provided by computer simulations

Protein-protein interaction site mapping using NMR-detected mutational scanning.

We demonstrate a novel NMR method for the mapping of protein-protein interaction sites. In our approach protein-protein binding sites are mapped by competition binding experiments using indirect NMR reporter technology and Ala positional scanning. The methodology provides high sensitivity, ease of implementation and high-throughput capabilities. The feasibility of the technique is demonstrated with an application to the beta-Catenin/Tcf4 complex

Mitochondrial differentiation in a polymorphic land snail : evidence for Pleistocene survival within the boundaries of permafrost

The genetic differentiation of populations having colonized formerly unsuitable habitats after the Pleistocene glaciations depends to a great extent on the speed of expansion. Slow dispersers maintain their refugial diversity whereas fast dispersal leads to a reduction of diversity in the newly colonized areas. During the Pleistocene, almost the entire current range of the land snail Arianta arbustorum has repeatedly been covered with ice or been subjected to permafrost. Owing to the low potential for dispersal of land snails, slow (re)colonization of the wide range from southern refugia can be excluded. Alternatively, fast, passive dispersal from southern refugia or survival in and expansion from multiple refugia within the area subjected to permafrost may account for the current distribution. To distinguish between these scenarios we reconstructed a phylogeography based on the sequences of a fragment of the cytochrome oxidase I from 133 individuals collected at 45 localities and analysed the molecular variance. Seventy-five haplotypes were found that diverged on average at 7.52 arbustorum is an old species in which the population structure, isolation and the hermaphroditic nature have reduced the probability of lineage extinction. The genetic structure was highly significant with the highest variance partition found among regions. Geographic distance and mitochondrial differentiation were not congruent. Lineages had overlapping ranges. The clear genetic differentiation and the patchy pattern of haplotype distribution suggest that colonization of formerly unsuitable habitats was mainly achieved from multiple populations from within the permafrost area