Rational Design of Far-Red Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators: from Elucidation of the Fluorescence Mechanism in Archers to Novel Red-Shifted Variants.

Genetically encoded voltage indicators (GEVIs) have found wide applications as molecular tools for visualization of changes in cell membrane potential. Among others, several classes of archaerhodopsin-3-based GEVIs have been developed and have proved themselves promising in various molecular imaging studies. To expand the application range for this type of GEVIs, new variants with absorption band maxima shifted toward the first biological window and enhanced fluorescence signal are required. Here, we integrate computational and experimental strategies to reveal structural factors that distinguish far-red bright archaerhodopsin-3-based GEVIs, Archers, obtained by directed evolution in a previous study (McIsaac et al., PNAS, 2014) and the wild-type archaerhodopsin-3 with an extremely dim fluorescence signal, aiming to use the obtained information in subsequent rational design. We found that the fluorescence can be enhanced by stabilization of a certain conformation of the protein, which, in turn, can be achieved by tuning the pK a value of two titratable residues. These findings were supported further by introducing mutations into wild-type archeorhodopsin-3 and detecting the enhancement of the fluorescence signal. Finally, we came up with a rational design and proposed previously unknown Archers variants with red-shifted absorption bands (λmax up to 640 nm) and potential-dependent bright fluorescence (quantum yield up to 0.97%).

Fluorescence of the Retinal Chromophore in Microbial and Animal Rhodopsins.

Fluorescence of the vast majority of natural opsin-based photoactive proteins is extremely low, in accordance with their functions that depend on efficient transduction of absorbed light energy. However, several recently proposed classes of engineered rhodopsins with enhanced fluorescence, along with the discovery of a new natural highly fluorescent rhodopsin, NeoR, opened a way to exploit these transmembrane proteins as fluorescent sensors and draw more attention to studies on this untypical rhodopsin property. Here, we review the available data on the fluorescence of the retinal chromophore in microbial and animal rhodopsins and their photocycle intermediates, as well as different isomers of the protonated retinal Schiff base in various solvents and the gas phase.

Development of Fluorescent Isocoumarin-Fused Oxacyclononyne - 1,2,3-Triazole Pairs.

Fluorescent isocoumarin-fused cycloalkynes, which are reactive in SPAAC and give fluorescent triazoles regardless of the azide nature, have been developed. The key structural feature that converts the non-fluorescent cycloalkyne/triazole pair to its fluorescent counterpart is the pi-acceptor group (COOMe, CN) at the C6 position of the isocoumarin ring. The design of the fluorescent cycloalkyne/triazole pairs is based on the theoretical study of the S1 state deactivation mechanism of the non-fluorescent isocoumarin-fused cycloalkyne IC9O using multi-configurational ab initio and DFT methodologies. The calculations revealed that deactivation proceeds through the electrocyclic ring opening of the α-pyrone cycle and is accompanied by a redistribution of electron density in the fused benzene ring. We proposed that the S1 excited state deactivation barrier could be increased by introducing a pi-acceptor group into a position that is in direct conjugation with the formed C=O group and has a reduced electron density in the transition state. As a proof of concept, we designed and synthesized two fluorescent isocoumarin-fused cycloalkynes IC9O-COOMe and IC9O-CN bearing pi-acceptors at the C6 position. The importance of the nature of a pi-acceptor group was shown by the example of much less fluorescent CF3 -substituted cycloalkyne IC9O-CF3 .

Fluorescence Imaging of Cell Membrane Potential: From Relative Changes to Absolute Values.

Membrane potential is a fundamental property of biological cells. Changes in membrane potential characterize a vast number of vital biological processes, such as the activity of neurons and cardiomyocytes, tumorogenesis, cell-cycle progression, etc. A common strategy to record membrane potential changes that occur in the process of interest is to utilize organic dyes or genetically-encoded voltage indicators with voltage-dependent fluorescence. Sensors are introduced into target cells, and alterations of fluorescence intensity are recorded with optical methods. Techniques that allow recording relative changes of membrane potential and do not take into account fluorescence alterations due to factors other than membrane voltage are already widely used in modern biological and biomedical studies. Such techniques have been reviewed previously in many works. However, in order to investigate a number of processes, especially long-term processes, the measured signal must be corrected to exclude the contribution from voltage-independent factors or even absolute values of cell membrane potential have to be evaluated. Techniques that enable such measurements are the subject of this review.

Heterocycloalkynes Fused to a Heterocyclic Core: Searching for an Island with Optimal Stability-Reactivity Balance.

In the search for fundamentally new, active, stable, and readily synthetically accessible cycloalkynes as strain-promoted azide-alkyne cycloaddition (SPAAC) reagents for bioorthogonal bioconjugation, we integrated two common approaches: the reagent destabilization by the increase of a ring strain and the transition state stabilization through electronic effects. As a result new SPAAC reagents, heterocyclononynes fused to a heterocyclic core, were created. These compounds can be obtained through a general synthetic route based on four crucial steps: the electrophile-promoted cyclization, Sonogashira coupling, Nicholas reaction, and final deprotection of Co-complexes of cycloalkynes from cobalt. Varying the natures of the heterocycle and heteroatom allows for reaching the optimal stability-reactivity balance for new strained systems. Computational and experimental studies revealed similar SPAAC reactivities for stable 9-membered isocoumarin- and benzothiophene-fused heterocycloalkynes and their unstable 8-membered homologues. We discovered that close reactivity is a result of the interplay of two electronic effects, which stabilize SPAAC transition states (πin* → σ* and π* → πin*) with structural effects such as conformational changes from eclipsed to staggered conformations in the cycloalkyne scaffold, that noticeably impact alkyne bending and reactivity. The concerted influence of a heterocycle and a heteroatom on the polarization of a triple bond in highly strained cycles along with a low HOMO-LUMO gap was assumed to be the reason for the unpredictable kinetic instability of all the cyclooctynes and the benzothiophene-fused oxacyclononyne. The applicability of stable isocoumarin-fused azacyclononyne IC9N-BDP-FL for in vitro bioconjugation was exemplified by labeling and visualization of HEK293 cells carrying azido-DNA and azido-glycans.

Free Energy Computation for an Isomerizing Chromophore in a Molecular Cavity via the Average Solvent Electrostatic Configuration Model: Applications in Rhodopsin and Rhodopsin-Mimicking Systems.

We present a novel technique for computing the free energy differences between two chromophore "isomers" hosted in a molecular environment (a generalized solvent). Such an environment may range from a relatively rigid protein cavity to a flexible solvent environment. The technique is characterized by the application of the previously reported "average electrostatic solvent configuration" method, and it is based on the idea of using the free energy perturbation theory along with a chromophore annihilation procedure in thermodynamic cycle calculations. The method is benchmarked by computing the ground-state room-temperature relative stabilities between (i) the cis and trans isomers of prototypal animal and microbial rhodopsins and (ii) the analogue isomers of a rhodopsin-like light-driven molecular switch in methanol. Furthermore, we show that the same technology can be used to estimate the activation free energy for the thermal isomerization of systems i-ii by replacing one isomer with a transition state. The results show that the computed relative stability and isomerization barrier magnitudes for the selected systems are in line with the available experimental observation in spite of their widely diverse complexity.

Simple Models to Study Spectral Properties of Microbial and Animal Rhodopsins: Evaluation of the Electrostatic Effect of Charged and Polar Residues on the First Absorption Band Maxima.

A typical feature of proteins from the rhodopsin family is the sensitivity of their absorption band maximum to protein amino acid composition. For this reason, studies of these proteins often require methodologies that determine spectral shift caused by amino acid substitutions. Generally, quantum mechanics/molecular mechanics models allow for the calculation of a substitution-induced spectral shift with high accuracy, but their application is not always easy and requires special knowledge. In the present study, we propose simple models that allow us to estimate the direct effect of a charged or polar residue substitution without extensive calculations using only rhodopsin three-dimensional structure and plots or tables that are provided in this article. The models are based on absorption maximum values calculated at the SORCI+Q level of theory for cis- and trans-forms of retinal protonated Schiff base in an external electrostatic field of charges and dipoles. Each value corresponds to a certain position of a charged or polar residue relative to the retinal chromophore. The proposed approach was evaluated against an example set consisting of twelve bovine rhodopsin and sodium pumping rhodopsin mutants. The limits of the applicability of the models are also discussed. The results of our study can be useful for the interpretation of experimental data and for the rational design of rhodopsins with required spectral properties.

An assessment of water placement algorithms in quantum mechanics/molecular mechanics modeling: the case of rhodopsins' first spectral absorption band maxima.

Quantum mechanics/molecular mechanics (QM/MM) models are a widely used tool to obtain detailed insight into the properties and functioning of proteins. The outcome of QM/MM studies heavily depends on the quality of the applied QM/MM model. Prediction and right placement of internal water molecules in protein cavities is one of the critical parts of any QM/MM model construction. Herein, we performed a systematic study of four protein hydration algorithms. We tested these algorithms for their ability to predict X-ray-resolved water molecules for a set of membrane photosensitive rhodopsin proteins, as well as the influence of the applied water placement algorithms on the QM/MM calculated absorption maxima (λmax) of these proteins. We used 49 rhodopsins and their intermediates with available X-ray structures as the test set. We found that a proper choice of hydration algorithms and setups is needed to predict functionally important water molecules in the chromophore-binding cavity of rhodopsins, such as the water cluster in the N-H region of bacteriorhodopsin or two water molecules in the binding pocket of bovine visual rhodopsin. The QM/MM calculated λmax of rhodopsins is also quite sensitive to the applied protein hydration protocols. The best methodology allows obtaining an 18.0 nm average value for the absolute deviation of the calculated λmax from the experimental λmax. Although the major effect of water molecules on λmax originates from the water molecules located in the binding pocket, the water molecules outside the binding pocket also affect the calculated λmax mainly by causing a reorganization of the protein structure. The results reported in this study can be used for the evaluation and further development of hydration methodologies, in general, and rhodopsin QM/MM models, in particular.

A Comparative Study of Modern Homology Modeling Algorithms for Rhodopsin Structure Prediction.

Rhodopsins are seven α-helical membrane proteins that are of great importance in chemistry, biology, and modern biotechnology. Any in silico study on rhodopsin properties and functioning requires a high-quality three-dimensional structure. Due to particular difficulties with obtaining membrane protein structures from the experiment, in silico prediction of the three-dimensional rhodopsin structure based only on its primary sequence is an especially important task. For the last few years, significant progress was made in the field of protein structure prediction, especially for methods based on comparative modeling. However, the majority of this progress was made for soluble proteins and further investigations are needed to achieve similar progress for membrane proteins. In this paper, we evaluate the performance of modern protein structure prediction methodologies (implemented in the Medeller, I-TASSER, and Rosetta packages) for their ability to predict rhodopsin structures. Three widely used methodologies were considered: two general methodologies that are commonly applied to soluble proteins and a methodology that uses constraints that are specific for membrane proteins. The test pool consisted of 36 target-template pairs with different sequence similarities that was constructed on the basis of 24 experimental rhodopsin structures taken from the RCSB database. As a result, we showed that all three considered methodologies allow obtaining rhodopsin structures with the quality that is close to the crystallographic one (root mean square deviation (RMSD) of the predicted structure from the corresponding X-ray structure up to 1.5 Å) if the target-template sequence identity is higher than 40%. Moreover, all considered methodologies provided structures of average quality (RMSD < 4.0 Å) if the target-template sequence identity is higher than 20%. Such structures can be subsequently used for further investigation of molecular mechanisms of protein functioning and for the development of modern protein-based biotechnologies.