Exploration of natural red-shifted rhodopsins using a machine learning-based Bayesian experimental design

Abstract

31Microbial rhodopsins are photoreceptive membrane proteins utilized as molecular tools in 32 optogenetics. In this paper, a machine learning (ML)-based model was constructed to 33 approximate the relationship between amino acid sequences and absorption wavelengths using 34 ∼800 rhodopsins with known absorption wavelengths. This ML-based model was specifically 35 designed for screening rhodopsins that are red-shifted from representative rhodopsins in the 36 same subfamily. Among 5,558 candidate rhodopsins suggested by a protein BLAST search of 37 several protein databases, 40 were selected by the ML-based model. The wavelengths of these 38 40 selected candidates were experimentally investigated, and 32 (80%) showed red-shift gains. 39 In addition, four showed red-shift gains > 20 nm, and two were found to have desirable ion-40 transporting properties, indicating that they were potentially useful in optogenetics. These 41 findings suggest that an ML-based model can reduce the cost for exploring new functional 42 proteins. 43 44 48 (Fig. 1a). The first microbial rhodopsin, bacteriorhodopsin (BR), was discovered in the plasma 49 membrane of the halophilic archaea Halobacterium salinarum (formerly called H. halobium) 2 . 50 BR forms a purple-coloured patch in the plasma membrane called purple membrane, which 51 outwardly transports H + using sunlight energy 3 . After the discovery of BR, various types of 52 microbial rhodopsins were reported from diverse microorganisms, and recent progress in 53 genome sequencing techniques has uncovered several thousand microbial rhodopsin genes 1,4-54 6 . These microbial rhodopsins show various types of biological functions upon light absorption, 55 leading to all-trans-to-13-cis retinal isomerization. Among these, ion transporters, including 56 light-driven ion pumps and light-gated ion channels, are the most ubiquitous (Fig. 1b). Ion-57 transporting rhodopsins can transport several types of cations and anions, including H + , Na + , 58 K + , halides (Cl -, Br -, I -), NO -, and SO4 2-1,7-9 . The molecular mechanisms of ion-transporting 59 rhodopsins have been detailed in numerous biophysical, structural, and theoretical studies 1 . 60 In recent years, many ion-transporting rhodopsins have been used as molecular tools in 61 optogenetics to control the activity of animal neurons optically in vivo by heterologous 62 expression 10 , and optogenetics has revealed various new insights regarding the neural network 63 relevant to memory, movement, and emotional behaviour 11-14 . However, strong light scattering 64 by biological tissues and the cellular toxicity of shorter wavelength light make precise optical 65 control difficult. To circumvent this difficulty, new molecular optogenetics tools based on red-66shifted rhodopsins that can be controlled by weak scattering and low toxicity longer-67 wavelength light are urgently needed. Therefore, many approaches to obtain red-shifted 68 rhodopsins, including gene screening, amino acid mutation based on biophysical and structural 69 ...

PhISCO: a simple method to infer phenotypes from protein sequences

“…Based on a previous work by Karasuma et al [8] and Inoue et al [24] we evaluated whether our method was able to identify positions associated with the fine tuning of the absorptionwavelength of microbial rhodopsins. To this end, we worked with the published data containing microbial rhodopsin sequences and associated wavelengths [8].…”