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A Voltage-Dependent Fluorescent Indicator for Optogenetic Applications, Archaerhodopsin-3: Structure and Optical Properties

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A Voltage-Dependent Fluorescent Indicator for Optogenetic Applications, Archaerhodopsin-3: Structure and Optical Properties F1000Research 2017, 6:33 Last updated: 30 JUL 2021 RESEARCH NOTE A voltage-dependent fluorescent indicator for optogenetic applications, archaerhodopsin-3: Structure and optical properties from in silico modeling [version 3; peer review: 3 approved] Dmitrii M. Nikolaev1, Anton Emelyanov1, Vitaly M. Boitsov1, Maxim S Panov2, Mikhail N. Ryazantsev 2,3 1Saint-Petersburg National Research Academic University of the Russian Academy of Science, St. Petersburg, Russian Federation 2Saint-Petersburg State University, St. Petersburg, Russian Federation 3Saint-Petersburg Scientific Center of the Russian Academy of Sciences, St. Petersburg, Russian Federation v3 First published: 11 Jan 2017, 6:33 Open Peer Review https://doi.org/10.12688/f1000research.10541.1 Second version: 17 Jan 2017, 6:33 https://doi.org/10.12688/f1000research.10541.2 Reviewer Status Latest published: 15 Nov 2017, 6:33 https://doi.org/10.12688/f1000research.10541.3 Invited Reviewers 1 2 3 Abstract It was demonstrated in recent studies that some rhodopsins can be version 3 used in optogenetics as fluorescent indicators of membrane voltage. (revision) report report One of the promising candidates for these applications is 15 Nov 2017 archaerhodopsin-3. While it has already shown encouraging results, there is still a large room for improvement. One of possible directions version 2 is increasing the intensity of the protein's fluorescent signal. Rational (revision) report report report design of mutants with an improved signal is an important task, which 17 Jan 2017 requires both experimental and theoretical studies. Herein, we used a homology-based computational approach to predict the three- version 1 dimensional structure of archaerhodopsin-3, and a Quantum 11 Jan 2017 Mechanics/Molecular Mechanics (QM/MM) hybrid approach with high- level multireference ab initio methodology (SORCI+Q/AMBER) to 1. Kirill A. Afonin, The University of North model optical properties of this protein. We demonstrated that this Carolina at Charlotte, Charlotte, USA methodology allows for reliable prediction of structure and spectral properties of archaerhodopsin-3. The results of this study can be 2. Adam E. Cohen, Harvard University, utilized for computational molecular design of efficient fluorescent Cambridge, USA indicators of membrane voltage for modern optogenetics on the basis Samouil Farhi, Harvard University, of archaerhodopsin-3. Cambridge, USA Keywords optogenetics, archaerhodopsin, protein structure prediction, QM/MM, 3. Jeremy N. Harvey, KU Leuven, Leuven, spectral tuning in rhodopsins Belgium Page 1 of 17 F1000Research 2017, 6:33 Last updated: 30 JUL 2021 Any reports and responses or comments on the article can be found at the end of the article. Corresponding author: Mikhail N. Ryazantsev ([email protected]) Author roles: Nikolaev DM: Investigation, Writing – Original Draft Preparation; Emelyanov A: Investigation, Writing – Original Draft Preparation; Boitsov VM: Investigation; Panov MS: Formal Analysis, Investigation; Ryazantsev MN: Investigation, Project Administration, Supervision, Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: MNR were supported by Russian Foundation for Basic Research (grant numbers, 14-04-01339 A, 15-29-03872 ofi_m and 16-04-00494 A). The work was funded by Ministry of Education and Science of Russian Federation (grant 16.9790.2017/BCh). The work was funded by a grant from the Presidium of the Russian Academy of Sciences. Copyright: © 2017 Nikolaev DM et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Nikolaev DM, Emelyanov A, Boitsov VM et al. A voltage-dependent fluorescent indicator for optogenetic applications, archaerhodopsin-3: Structure and optical properties from in silico modeling [version 3; peer review: 3 approved] F1000Research 2017, 6:33 https://doi.org/10.12688/f1000research.10541.3 First published: 11 Jan 2017, 6:33 https://doi.org/10.12688/f1000research.10541.1 Page 2 of 17 F1000Research 2017, 6:33 Last updated: 30 JUL 2021 prediction algorithm is not straightforward, we tested several REVISED Amendments from Version 2 methods. To evaluate the quality of obtained structures we per- Additional information and data analysis were added in Results formed subsequent Quantum Mechanics/Molecular Mechanics section of the article: 1. The comparison of results of different (QM/MM) calculations of absorption maxima and compared the sequence alignment algorithms (new Figure 1); 2. The results with available experimental data. comparison of the obtained model of archaerhodopsin-3 with the crystallographic structure of archaerhodopsin-1 (new Figure 2, Figure 3); 3. Analysis of the results of absorption maxima Methods calculations for archaerhodopsin-3. Information about analogous The structure of archaerhodopsin-3 was built using a homol- results for other rhodopsins is provided. 4. Residues replaced in ogy modeling approach. Primary structures for all rhodopsins site-directed mutagenesis study (Mclsaac RS et al., PNAS, 2014) were visualized (new Figure 4). with crystallographic data are available in the Protein Data Bank library6 (24 structures as of September 2016) were compared with Modifications made in the Introduction section and Abstract, the primary structure of archaerhodopsin-3. Archaerhodopsin-1, considering the remarks of our referees. In the Methods section information about sequence identity between archaerhodopsin-1 which has the highest sequence identity to the target protein, and archaerhodopsin-3 is provided. was chosen as a template (RCSB code 1UAZ, sequence identity Also, the new variant of the archieve with input and output files, 84.5%, sequence similarity 91.5%). Three algorithms of extra grant information (Russian Foundation for Basic Research homology-based model building were tested: Medeller7, and Presidium of the RAS) and acknowledgements to two I-TASSER8 and RosettaCM9. All methods of homology modeling computational research facilities (HPC computing resources at heavily rely on externally made target-template alignment of pri- Lomonosov Moscow State University and Computer Center of mary sequences, which serves as the main instruction for model SPbU) are provided. building. For this reason, we tested three algorithms of pairwise The additional grants listed, have aided in the continuation of the alignment using their results as an input for each method of model project that was supported by previous funding. building. Two of the alignment methods are specifically constructed See referee reports for membrane proteins, MP-T10 and AlignMe11, and the third one, MUSTER12, gains its quality from evolutionary predictions. The latter algorithm is a built-in algorithm of I-TASSER suite and its results were used only for this method of structure prediction. Introduction Precise and quick control of physiological processes using integrated Before QM/MM calculations, several preparation steps were optical and genetic methods is a vast area with a high number of performed: hydrogen atoms were added using pdb2pqr package13 important applications1. One possible approach in this field is using version 2.1.1 using CHARMM force field version 2714, pH=7; fluorescent voltage-dependent indicators for detecting the activity hydrogen atoms were equilibrated by energy minimization in of mammalian neurons, which allows achievement of the precision NAMD package15, version 2.11. The retinal chromophore was of a single neuron without perceptible time delays. It was recently bound to the lysine residue Lys226, whole lysine + retinal system shown that some rhodopsins, especially achaerhodopsin-3, can be was parameterized in CHARMM force field16. The protein was potential candidates for such a task2–5. While the results obtained inserted in the POPC membrane17, the whole system was inserted for archaerhodopsin-3 are already very encouraging, increasing the in a water solvent box, the TIP3P water model18 was used, the fluorescent signal of such proteins can lead to a significant progress size of water box was selected so that there were at least 10 Å from in this field. It was also shown that the insertion of mutations any atom of protein to the edge of the system, and the system was into these proteins can dramatically improve the signal quality2–5. neutralized by addition of Na+ and Cl- ions. Unfortunately, the fundamental mechanisms underlying the proc- Relaxation of the system was performed in several steps: esses that determine fluorescence are not well understood. This lack relaxation of retinal + lysine complex with all other atoms fixed, of knowledge leads to difficulties with design of desired rhodopsin relaxation of all atoms that were within 6 Å of the chromophore mutants. While rational design is not based on a solid foundation system, relaxation of whole protein and water box. During all and, for this reason, is not very effective, another experimental these steps the following parameters were used: a 10 Å cutoff with approach, random mutagenesis, is very time consuming. Computa- switching starting at 8.5 Å was applied to the electrostatics and tional studies can provide additional insights into the problem. One van der Waals interactions; Particle Mesh Ewald method19 was of the main obstacles for computational modeling of proteins is the used for dealing
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