Bioorthogonal catalytic activation of Platinum and Ruthenium anticancer complexes by FAD and flavoproteins

Recent advances in bioorthogonal catalysis promise to deliver new chemical tools for performing chemoselective transformations in complex biological environments. Herein, we report how FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), and four flavoproteins act as unconventional photocatalysts capable of converting PtIV and RuII complexes into potentially toxic PtII or RuII-OH2 species. In the presence of electron donors and low doses of visible light, the flavoproteins mini singlet oxygen generator (miniSOG) and NADH oxidase (NOX) catalytically activate PtIV prodrugs with bioorthogonal selectivity. In the presence of NADH, NOX catalyzes PtIV activation in the dark as well, indicating for the first time that flavoenzymes may contribute to initiating the activity of PtIV chemotherapeutic agents

Assessing the potential of photosensitizing flavoproteins as tags for correlative microscopy

Photosensitizing flavoproteins have great potential as tags for correlative light and electron microscopy (CLEM). We examine the photostability of miniSOG mutants and their ability to photo-oxidize diaminobenzidine, both key aspects for CLEM. Our experiments reveal a complex relation between these parameters and the production of different reactive oxygen species. ¸ 2016 The Royal Society of Chemistry

Correlative 3D cryo X-ray imaging reveals intracellular location and effect of designed antifibrotic protein-nanomaterial hybrids

Revealing the intracellular location of novel therapeutic agents is paramount for the understanding of their effect at the cell ultrastructure level. Here, we apply a novel correlative cryo 3D imaging approach to determine the intracellular fate of a designed protein-nanomaterial hybrid with antifibrotic properties that shows great promise in mitigating myocardial fibrosis. Cryo 3D structured illumination microscopy (cryo-3D-SIM) pinpoints the location and cryo soft X-ray tomography (cryo-SXT) reveals the ultrastructural environment and subcellular localization of this nanomaterial with spatial correlation accuracy down to 70 nm in whole cells. This novel high resolution 3D cryo correlative approach unambiguously locates the nanomaterial after overnight treatment within multivesicular bodies which have been associated with endosomal trafficking events by confocal microscopy. Moreover, this approach allows assessing the cellular response towards the treatment by evaluating the morphological changes induced. This is especially relevant for the future usage of nanoformulations in clinical practices. This correlative super-resolution and X-ray imaging strategy joins high specificity, by the use of fluorescence, with high spatial resolution at 30 nm (half pitch) provided by cryo-SXT in whole cells, without the need of staining or fixation, and can be of particular benefit to locate specific molecules in the native cellular environment in bio-nanomedicine

Repeat proteins as versatile scaffolds for arrays of redox-active FeS clusters

Arrays of one, two and four electron-transfer active [4Fe–4S] clusters were constructed on modular tetratricopeptide repeat protein scaffolds, with the number of clusters determined solely by the size of the scaffold. The constructs show reversible redox activity and transient charge stabilization necessary to facilitate charge transfer.This work has been supported by the European Research Council ERC-2014-CoG-648071-ProNANO (ALC), the Spanish Ministry of Economy and Competitiveness (MINECO) BIO2016-77367-C2-1-R (ALC), the Basque Government Elkartek KK-2017/00008 and the National Science Foundation (CHE-CLP award SusChEM 1508301). This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency – Grant No. MDM-2017-0720. SHM thanks the Basque Government for financial support through a PhD fellowship

Repeat protein scaffolds: Ordering photo- and electroactive molecules in solution and solid state

The precise control over the organization of photoactive components at the nanoscale is one of the main challenges for the generation of new and sophisticated macroscopically ordered materials with enhanced properties. In this work we present a novel bioinspired approach using protein-based building blocks for the arrangement of photo- and electroactive porphyrin derivatives. We used a designed repeat protein scaffold with demonstrated unique features that allow for the control of their structure, functionality, and assembly. Our designed domains act as exact biomolecular templates to organize porphyrin molecules at the required distance. The hybrid conjugates retain the structure and assembly properties of the protein scaffold and display the spectroscopic features of orderly aggregated porphyrins along the protein structure. Finally, we achieved a solid ordered bio-organic hybrid thin film with anisotropic photoconductivity. ¸ 2016 The Royal Society of Chemistry.This work has been supported by the European Commission IRG-246688 Bionanotools (ALC), the Spanish Ministry of Economy and Competitiveness (MINECO) BIO2012-34835 (ALC) and CTQ2014-520456-R (NM) and the European Research Council ERC-320441-Chirallcarbon (NM), and ERC-2014-CoG-648071 (ProNANO) (ALC). C. A. thanks to the Ramón y Cajal granted and J. L.-A. thanks to Spanish Ministry of Education for FPU granted. SHM thanks the Basque Government for financial support through a PhD fellowship. KPE gives thanks for financial support from the MIT–Spain internship program