@article{1611, abstract = {Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding "core" that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an l-arginine biosensor. l-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a Förster resonance energy transfer (FRET) biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a Kd of ∼14 μM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate l-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report l-arginine concentrations in brain tissue.}, author = {Whitfield, Jason and Zhang, William and Herde, Michel and Clifton, Ben and Radziejewski, Johanna and Janovjak, Harald L and Henneberger, Christian and Jackson, Colin}, journal = {Protein Science}, number = {9}, pages = {1412 -- 1422}, publisher = {Wiley}, title = {{Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction}}, doi = {10.1002/pro.2721}, volume = {24}, year = {2015}, } @article{1867, abstract = {Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools.}, author = {Hühner, Jens and Inglés Prieto, Álvaro and Neusüß, Christian and Lämmerhofer, Michael and Janovjak, Harald L}, journal = {Electrophoresis}, number = {4}, pages = {518 -- 525}, publisher = {Wiley}, title = {{Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection}}, doi = {10.1002/elps.201400451}, volume = {36}, year = {2015}, } @article{1678, abstract = {High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.}, author = {Inglés Prieto, Álvaro and Gschaider-Reichhart, Eva and Muellner, Markus and Nowak, Matthias and Nijman, Sebastian and Grusch, Michael and Janovjak, Harald L}, journal = {Nature Chemical Biology}, number = {12}, pages = {952 -- 954}, publisher = {Nature Publishing Group}, title = {{Light-assisted small-molecule screening against protein kinases}}, doi = {10.1038/nchembio.1933}, volume = {11}, year = {2015}, } @article{1844, abstract = {Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.}, author = {Risso, Valeria and Manssour Triedo, Fadia and Delgado Delgado, Asuncion and Arco, Rocio and Barroso Deljesús, Alicia and Inglés Prieto, Álvaro and Godoy Ruiz, Raquel and Gavira, Josè and Gaucher, Eric and Ibarra Molero, Beatriz and Sánchez Ruiz, Jose}, journal = {Molecular Biology and Evolution}, number = {2}, pages = {440 -- 455}, publisher = {Oxford University Press}, title = {{Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history}}, doi = {10.1093/molbev/msu312}, volume = {32}, year = {2014}, } @article{2032, abstract = {As light-based control of fundamental signaling pathways is becoming a reality, the field of optogenetics is rapidly moving beyond neuroscience. We have recently developed receptor tyrosine kinases that are activated by light and control cell proliferation, epithelial–mesenchymal transition, and angiogenic sprouting—cell behaviors central to cancer progression.}, author = {Inglés Prieto, Álvaro and Gschaider-Reichhart, Eva and Schelch, Karin and Janovjak, Harald L and Grusch, Michael}, journal = {Molecular and Cellular Oncology}, number = {4}, publisher = {Taylor & Francis}, title = {{The optogenetic promise for oncology: Episode I}}, doi = {10.4161/23723548.2014.964045}, volume = {1}, year = {2014}, }