Liubov Kost A, Ivanova V O, Ruchkin D A, Kolesov D V, Pletneva N V, Lukyanov K A, Nikitin E S and Bogdanov A M
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry-RAS, Russia
Institute of Higher Nervous Activity-RAS, Russia
Skolkovo Institute of Science and Technology, Russia
Scientific Tracks Abstracts: J Biosens Bioelectron
Genetically encoded voltage indicators (GEVIs) represent a promising tool for the neurophysiology experiments.
Namely, they would become a probe of choice for electric activity monitoring from the specifically targeted
cell populations or even at the live organism level. However, GEVIs practical characteristics remain suboptimal for
in vivo-studies and are being permanently improved. GEVI molecule usually consists of the three structural units:
voltage-sensing domain, fluorescent protein and amino acid linker between them. Generally, each of the mentioned
units can determine the performance of a particular indicator, and thus engineering of the improved GEVI could
imply the modification of any part of it. Here we use two parallel strategies to enhance VSD-FR189-188, recently
described red fluorescent protein-based voltage indicator. On one hand, we have modified amino acid content and
length of the linker which connects Ci-VSP voltage-sensing domain with the FusionRed-derived fluorescent unit.
Our experiments revealed that some variants with shorter linker demonstrated dramatically enhanced (up to 30-
fold) responsiveness relative to the parental GEVI, though almost unchanged or slightly decreased dynamic range.
To address the modest dynamic range issue, we implemented the second strategy implying the fluorescent core
modification. FusionRed protein, which forms VSD-FR189-188 fluorescent core, is known to have a partially cleaved
polypeptide chain. We supposed that the chain break might decrease the chromophore conformational sensitivity
within the GEVI construct thus limiting response dynamic range. X-ray data on the FusionRed structure obtained
recently point to a key role of Cys158 residue in the bond cleavage. Based on this hypothesis, we designed FusionRed
mutants (C158L, A157/C158L) which remain spectrally similar to the original protein but didnâ??t show chain cleavage.
These mutants are of interest as new fluorescent units for GEVIs.
Recent Publications:
1. Kost, L A, E S Nikitin, V O Ivanova, U Sung, E V Putintseva, D M Chudakov, P M Balaban, K A Lukyanov,
and A M Bogdanov (2017) “Insertion of the voltage-sensitive domain into circularly permuted red fluorescent
protein as a design for genetically encoded voltage sensor. PLoS ONE 12:9.
2. Shemiakina I I, G V Ermakova, P J Cranfill, M A Baird, R A Evans, E A Souslova, D B Staroverov, et al. (2012)
A monomeric red fluorescent protein with low cytotoxicity. Nature Communications 3(1):1204.
Liubov Kost A has completed her Graduation from Pirogov Russian National Research Medical University, Faculty of Biomedicine, Department of Medical Biophysics in 2014. Currently, she is a pursuing her PhD in Molecular Biology at Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow. Her research interests include the development of the enhanced fluorescent proteins and genetically encoded sensors based on them.
E-mail: lu.kurkova@gmail.com
Biosensors & Bioelectronics received 1751 citations as per Google Scholar report