Single-molecule fret study on structural dynamics of membrane proteins

De Na+ -gekoppelde betaïne symporter BetP van de bacterie Corynebacterium glutamicum gaat hyperosmotische stress tegen en wordt gereguleerd via een osmodetecterend C-terminaal domein. Hoewel de biochemie van het systeem goed is gekarakteriseerd, is structurele informatie slechts gedeeltelijk beschikbaar, vanwege een ontbrekend C-terminaal domein in de kristalstructuur. Dus zowel het activerings- als transportmechanisme blijven onopgelost. We hebben verschillende enkele molecuul technieken ontwikkeld om de conformationele dynamiek en heterogeniteit van BetP te bestuderen in proof-of-principle studies en een gedetailleerde mechanistische studie van transportactivatie.The Na+ coupled betaine symporter BetP counteracts hyperosmotic stress and is regulated via an osmosensing C-terminal domain. While the biochemistry of the system is well characterized, structural information is only partially available due to a lacking C-terminal domain in the crystal structure. Thus, both the activation and transport mechanism remain elusive. In this work, we established different single-molecule-based methods to study the conformational dynamics and heterogeneity of BetP in proof-of-principle studies (chapter 2/3) and a detailed mechanistic study of transport activation. Our studies showed that smFRET approach was sussefully adopted for BetP and we obtained the results that was not yet accompolished by standard strucrtal biology teqniques such as crystal structure method. The established methods will pave the way for future single-molecule studies and enhanced mechanistic understanding of the secondary-active transporter BetP in a near-native biochemistry environment

Caging and Photoactivation in Single-Molecule Forster Resonance Energy Transfer Experiments

Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Forster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor acceptor pairs during solution-based smFRET. We use this "caged FRET" methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules

A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization

Intramolecular photostabilization via triple-state quenching was recently revived as a tool to impart synthetic organic fluorophores with 'self-healing' properties. To date, utilization of such fluorophore derivatives is rare due to their elaborate multi-step synthesis. Here we present a general strategy to covalently link a synthetic organic fluorophore simultaneously to a photostabilizer and biomolecular target via unnatural amino acids. The modular approach uses commercially available starting materials and simple chemical transformations. The resulting photostabilizer-dye conjugates are based on rhodamines, carbopyronines and cyanines with excellent photophysical properties, that is, high photostability and minimal signal fluctuations. Their versatile use is demonstrated by single-step labelling of DNA, antibodies and proteins, as well as applications in single-molecule and super-resolution fluorescence microscopy. We are convinced that the presented scaffolding strategy and the improved characteristics of the conjugates in applications will trigger the broader use of intramolecular photostabilization and help to emerge this approach as a new gold standard

Author Correction: A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization

Nature Communications 7: Article number: 10144 (2016); Published 11 January 2016, Updated 24 July 2018 The original version of this Article omitted the following from the Acknowledgements: ‘This work was also financed by an ERC starting grant ‘SM-IMPORT’ (No. 638536 to T.C.)’. This has been corrected in both the PDF and HTML versions of the Article.</p