Graphene Oxide Modulating the Bioelectronic Properties of Penicillinase Immobilized in Lipid Langmuir-Blodgett Films.

In this paper, graphene oxide was incorporated in penicillinase-lipid Langmuir monolayers and transferred to solid supports as Langmuir-Blodgett (LB) films so that the enzyme catalytic properties could be evaluated. Adsorption of penicillinase and graphene oxide on dimyristoylphosphatidic acid (DMPA) monolayers at the air-water interface was investigated by tensiometry, vibrational spectroscopy, and Brewster angle microscopy. The LB films were characterized by quartz crystal microbalance, infrared spectroscopy, luminescence spectroscopy, and atomic force microscopy. Enzyme activity was studied with UV-vis spectroscopy, and the feasibility of the supramolecular device nanostructured as ultrathin films was essayed as an optical sensor device. The presence of graphene oxide in the enzyme-lipid LB film not only tuned the catalytic activity of penicillinase but also helped conserve its enzyme activity after weeks. These results may be related not only to the molecular architecture provided by the film but also to the synergism between the compounds on the active layer, leading to a molecular architecture that allowed a fast analyte diffusion owing to a suitable molecular accommodation which also preserved the penicillinase activity. This work then demonstrates the feasibility of employing LB films composed of lipids, graphene oxide, and enzymes as optical devices for biosensing applications as a proof-of-concept experiment.

Opening of a cryptic pocket in ß-lactamase increases penicillinase activity.

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in ß-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across ß-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM ß-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket's functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

Comparative activity of beta-lactam agents (carbapenem excepted) against Pseudomonas aeruginosa strains with CARB or OXA beta-lactamases.

BACKGROUND: Ticarcillin resistance in Pseudomonas aeruginosa can be mediated by various beta-lactamases of the CARB and OXA groups. METHODS: We investigated the activity of seven beta-lactam agents and two beta-lactam-beta-lactamase inhibitor combinations against 216 P.aeruginosa strains with genotypically characterized beta-lactamases, including 137 CARB, 31 OXA-35, 25 OXA-10, 13 OXA-1 and 10 OXA-2. MICs were determined by the agar dilution method. RESULTS: The activities of ticarcillin and piperacillin were more affected by CARB than by OXA enzymes. beta-Lactamase inhibitors were poorly effective against OXA-1, OXA-35 and OXA-10. OXA-1 conferred resistance to cefepime and cefpirome but not to cefsulodin and aztreonam. Ceftazidime remained the most active agent against all groups of enzymes. Major differences in the susceptibility rates according to NCCLS and CASFM breakpoints were observed. CONCLUSIONS: Significant differences were found in the resistance profile associated with the various types of CARB and OXA beta-lactamases in P. aeruginosa.