Biology subjects

Wachtveitl, J.

Publications and source records attributed to Wachtveitl, J..

8 recordsLinked to original sources

bioRxiv2026

De novo design of light-controlled conformational protein switches

Light-responsive proteins offer the ability to control biology at an unmatched resolution. Yet, optogenetics is limited to a relatively small number of natural and engineered photoreceptors. The design of light-responsive proteins with native-like complexity is challenging as they must incorporate a light-responsive chromophore, couple chemical changes in the chromophore to structural changes in the protein, and ultimately propagate these changes to downstream effectors. Here, we present the computational design of new light-responsive proteins entirely from the bottom-up. We design de novo proteins that accurately bind a retinal chromophore and further design a protein conformational switching motion linked to retinal isomerisation. This switch unlocks a new protein-peptide interaction, creating light-activated heterodimers with an over 30-fold increase in binding upon illumination. Moreover, we show that the designed heterodimers are customisable, with tuneable wavelengths, sensitivities, and specificities. Further, we use the designed heterodimers to control biological function, regulating intracellular protein-protein interactions and cell-signalling receptors. Finally, we use real-time light-activated NMR to demonstrate that they undergo a defined light-activated conformational change. Overall, we computationally design a new class of light-responsive proteins that may open a new optogenetics toolbox with applications in biology.

biochemistry↗

bioRxiv2026

Computational design of microbial and animal rhodopsin soluble analogues

The computational design of soluble analogues of membrane proteins has unlocked exciting opportunities for the integration of unique membrane protein functions into soluble proteins. Here, we use AF2seq to generate accurate soluble analogues of both animal and microbial rhodopsins, based on the membrane GPCR topology, and the microbial rhodopsin transmembrane fold. We characterize the analogues and demonstrate that they are well-folded and highly thermostable. Furthermore, they exhibit the expected red shift characteristic of retinal binding. Top-down mass spectrometry confirms the placement of retinal covalent attachment, while X-ray crystallography validates the structural fidelity of the microbial rhodopsin analogue. Notably, the microbial rhodopsin analogue retains the primary reaction of the retinal photocycle, closely matching that of the native membrane protein. Overall, this work advances the possibility to transfer unique membrane protein functions, such as retinal photoswitching, into the soluble proteome.

Synthetic Biology↗

bioRxiv2025

A unique mechanism explaining the outstanding performance of a newly discovered doxycycline riboswitch

Synthetic riboswitches offer a versatile and protein-independent solution for conditional gene regulation. They consist of a regulatory domain and an aptamer domain that binds a specific ligand, with their performance largely depending on the ability of the aptamer to control the regulatory domain. Expanding the range of synthetic riboswitches therefore requires the discovery and characterization of new regulatory aptamers. In the present study, we identified a doxycycline-binding aptamer with outstanding regulatory properties in both yeast and human cells which are based on a unique structural dynamic upon ligand binding. Single-molecule force spectroscopy revealed that doxycycline binding strongly stabilizes an intermediate aptamer conformation offering mechanistic insights into its function. The identification of the aptamer through a combination of parallel SELEX and subsequent in vivo screening in yeast, also provided valuable insights into selection dynamics and the first proof for the effectiveness of RNA Capture-SELEX in aptamer selection. Together, the presented data deepen our understanding of regulatory aptamer selection and functionality while adding a high-performing doxycycline-responsive aptamer to the synthetic biology toolbox.

molecular biology↗

bioRxiv2025

Structural basis for no retinal binding in flotillin-associated rhodopsins

Rhodopsins are light-sensitive membrane proteins capturing solar energy via a retinal cofactor covalently attached to a lysine residue. Several groups of rhodopsins were reported to lack the conserved lysine and showed no retinal binding. Recently, flotillin-associated rhodopsins (FArhodopsins) were identified and suggested to lack the typical retinal binding pocket despite preserving the lysine residue in many members of the group. Here we present cryo-EM structures of paralog FArhodopsin and proteorhodopsin from marine bacteria Pseudothioglobus. The structures revealed pentameric assemblies of both proteins similar to those of other microbial rhodopsins. We demonstrate no binding of retinal to the FArhodopsin despite preservation of the lysine residue and overall similarity of the protein fold and internal organization to those of the retinal-binding paralog. Mutational analysis confirmed that two amino acids, H84 and E120, prevent retinal binding within the FArhodopsin. Thus, our work provides insights into the natural retinal loss in microbial rhodopsins and might contribute to the further understanding of the FArhodopsin clade.

biophysics↗

bioRxiv2024

CryoRhodopsins: a comprehensive characterization of a new clade of microbial rhodopsins from cold environments

Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin group from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have dual functionality switching between inward transmembrane proton translocation and photosensory activity, both of which can be modulated with UV light. CryoR1 exhibits two subpopulations in the ground state, which upon light activation lead to transient photocurrents of opposing polarities. A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by light, we demonstrate that the arginine stabilizes a UV-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified group and demonstrate principles of the adaptation of these rhodopsins to low temperatures.

biophysics↗

bioRxiv2023

A novel subgroup of light-driven sodium pumps with an additional Schiff base counterion

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identified a new subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterized a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and showed that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a new subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

biophysics↗

bioRxiv2022

Molecular snapshots of drug release from tubulin over eleven orders of magnitude in time

The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we captured the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.

molecular biology↗

bioRxiv2020

Temperature Dependence of the Krokinobacter rhodopsin 2 Kinetics

Here we applied target analysis to a temperature dependent flash photolysis dataset of the light-driven sodium ion pump Krokinobacter rhodopsin 2 (KR2) at sodium pumping conditions. With an increase in temperature from 10 - 40 {degrees}C, the overall photocycle duration was accelerated by a factor of six, while single transitions like the L to M transition increased by a factor of 40. Using kinetic modeling with the Eyring constraint as well as spectral corrections on the datasets the spectral position as well as the equilibria of the different photointermediates could be resolved. The results provide further insight into KR2s photocycle and energetics. STATEMENT OF SIGNIFICANCEKR2 is the most prominent member of the new class of non-proton cation pumps, as it represents an interesting new optogenetic tool. Despite widespread biophysical investigations, the molecular mechanisms of light-induced sodium pumping in KR2 are still not sufficiently understood. Therefore, an expanded set of thermodynamic parameters is essential for a complete picture. Our study of the KR2 photocycle shows that different steps in the photocycle are affected differently by temperature changes. Rigorous data analysis provides strong evidence that the transient states observed in time-resolved experiments represent rather equilibria between the different photocycle intermediates than pure intermediates. Gaining access to the dynamics and energetics of KR2 helps to answer long standing open questions concerning the molecular mechanism of cation pumping.

biophysics↗