Characterization and evaluation of photolabile (µ-peroxo)(µ-hydroxo)bis[bis(bipyridyl)cobalt caged oxygen compounds to facilitate time-resolved crystallographic studies of cytochrome c oxidase.

Photolabile (µ-peroxo)(µ-hydroxo)bis[bis(bipyridyl)-cobalt-based caged oxygen compounds have been synthesized and characterized by optical absorbance spectroscopy, X-ray crystallography. and the quantum yield and redox stability were investigated. Furthermore, conditions were established where redox incompatibilities encountered between caged oxygen compounds and oxygen-dependant cytochrome c oxidase (CcO) could be circumvented. Herein, we demonstrate that millimolar concentrations of molecular oxygen can be released from a caged oxygen compound with spatio-temporal control upon laser excitation, triggering enzymatic turnover in cytochrome c oxidase. Spectroscopic evidence confirms the attainment of a homogeneous reaction initiation at concentrations and conditions relevant for further crystallography studies. This was demonstrated by the oxidizing microcrystals of reduced CcO by liberation of millimolar concentrations of molecular oxygen from a caged oxygen compound. We believe this will expand the scope of available techniques for the detailed investigation of oxygen-dependant enzymes with its native substrate and facilitate further time-resolved X-ray based studies such as wide/small angle X-ray scattering and serial femtosecond crystallography.

No observable non-thermal effect of microwave radiation on the growth of microtubules.

Despite widespread public interest in the health impact of exposure to microwave radiation, studies of the influence of microwave radiation on biological samples are often inconclusive or contradictory. Here we examine the influence of microwave radiation of frequencies 3.5 GHz, 20 GHz and 29 GHz on the growth of microtubules, which are biological nanotubes that perform diverse functions in eukaryotic cells. Since microtubules are highly polar and can extend several micrometres in length, they are predicted to be sensitive to non-ionizing radiation. Moreover, it has been speculated that tubulin dimers within microtubules might rapidly toggle between different conformations, potentially participating in computational or other cooperative processes. Our data show that exposure to microwave radiation yields a microtubule growth curve that is distorted relative to control studies utilizing a homogeneous temperature jump. However, this apparent effect of non-ionizing radiation is reproduced by control experiments using an infrared laser or hot air to heat the sample and thereby mimic the thermal history of samples exposed to microwaves. As such, no non-thermal effects of microwave radiation on microtubule growth can be assigned. Our results highlight the need for appropriate control experiments in biophysical studies that may impact on the sphere of public interest.

Structural and biochemical analysis of family 92 carbohydrate-binding modules uncovers multivalent binding to ß-glucans.

Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to ß-1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation.