Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.

Yeast as a tool for membrane protein production and structure determination.

Membrane proteins are challenging targets to functionally and structurally characterize. An enduring bottleneck in their study is the reliable production of sufficient yields of stable protein. Here, we evaluate all eukaryotic membrane protein production experiments that have supported the deposition of a high-resolution structure. We focused on the most common yeast host systems, Saccharomyces cerevisiae and Pichia pastoris. The first high-resolution structure of a membrane protein produced in yeast was described in 1999 and today there are 186 structures of α-helical membrane proteins, representing 101 unique proteins from 37 families. Homologous and heterologous production are equally common in S. cerevisiae, while heterologous production dominates in P. pastoris, especially of human proteins, which represent about one-third of the total. Investigating protein engineering approaches (78 proteins from seven families) demonstrated that the majority contained a polyhistidine tag for purification, typically at the C-terminus of the protein. Codon optimization and truncation of hydrophilic extensions were also common approaches to improve yields. We conclude that yeast remains a useful production host for the study of α-helical membrane proteins.

Increased antibiotic efficacy and noninvasive monitoring of Staphylococcus epidermidis biofilms using per-cysteamine-substituted γ-cyclodextrin - A delivery effect validated by fluorescence microscopy.

Limited and poor delivery of antibiotics is cited as one reason for the difficulty in treating antibiotic-resistant biofilms associated with chronic infections. We investigate the effectiveness of a positively charged, single isomer cyclodextrin derivative, octakis[6-(2-aminoethylthio)-6-deoxy]-γ-CD (γCys) to improve the delivery of antibiotics to biofilms. Using multiphoton laser scanning microscopy complemented with super-resolution fluorescence microscopy, we showed that γCys tagged with fluorescein (FITC) is uniformly distributed throughout live S. epidermidis biofilm cultures in vitro and results suggest it is localized extracellularly in the biofilm matrix. NMR spectroscopic data in aqueous solution confirm that γCys forms inclusion complexes with both the antibiotics oxacillin and rifampicin. Efficacy of γCys/antibiotic (oxacillin and rifampicin) was measured in the biofilms. While treatment with γCys/oxacillin had little improvement over oxacillin alone, γCys/rifampicin reduced the biofilm viability to background levels demonstrating a remarkable improvement over rifampicin alone. The strong synergistic effect for γCys/rifampicin is at this stage not clearly understood, but plausible explanations are related to increased solubility of rifampicin upon complexation and/or synergistic interference with components of the biofilm. The results demonstrate that designed cyclodextrin nanocarriers, like γCys, efficiently deliver suitable antibiotics to biofilms and that fluorescence microscopy offers a novel approach for mechanistic investigations.