Biology subjects

Tassis, K.

Publications and source records attributed to Tassis, K..

3 recordsLinked to original sources

bioRxiv2020

Evolution of structural dynamics in bilobed proteins

Novel biophysical tools allow the structural dynamics of proteins, and the regulation of such dynamics by binding partners, to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remains poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the type-II periplasmic binding protein domain family, have undergone divergent evolution leading to modification of their structural dynamics and function. We performed a structural analysis of ~600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Forster resonance energy transfer and Hydrogen-Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enables distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extra-cytoplasmic transport-related proteins. Structural embellishments of the core created new interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately, altered substrate specificity. Our findings reveal an as yet unrecognized mechanism for the emergence of functional promiscuity during long periods of protein evolution and are applicable to a large number of domain architectures.

biophysics↗

bioRxiv2020

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Forster-resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the type-I ABC importer OpuA from Lactococcus lactis. We present data probing both intradomain distances that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC, and interdomain distances between SBDs or transmembrane domains. Using the methodology, we studied ligand-binding mechanisms as well as ATP and glycine betaine dependences of conformational changes. Our study expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

biophysics↗

bioRxiv2020

Characterization of fluorescent proteins with intramolecular photostabilization

Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Since there are no other natural fluorescent tags with comparable features, the impact of fluorescent proteins for biological research cannot be overemphasized. Despite their importance, their photophysical properties, i.e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties. The approach uses direct conjugation of photostablizing compounds such as triplet-state quenchers or redox-active substances to an organic fluorophore, thereby creating high local concentrations of photostabilizer. Here, we introduce an experimental strategy to screen for the effects of covalently-linked photostabilizers on fluorescent proteins. We recombinantly produced a double cysteine mutant (A206C/L221C) of -GFP for attachment of photostabilizer-maleimides on the {beta}-barrel in close proximity to the chromophore. Whereas labelling with photostabilizers such as Trolox, Nitrophenyl, and Cyclooctatetraene, which are often used for organic fluorophores, had no effect on -GFP-photostability, a substantial increase of photostability was found upon conjugation of -GFP to an azobenzene derivative. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the near-UV and blue spectral range. Our study paves the way towards the development and design of a second generation of fluorescent proteins with photostabilizers placed directly in the protein barrel by methods such as unnatural amino acid incorporation.

biophysics↗