Natural and Engineered Photoactivated Nucleotidyl Cyclases for Optogenetic Applications

Abstract

Cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers that regulate metabolic and behavioral responses in diverse organisms. We describe purification, engineering, and characterization of photoactivated nucleotidyl cyclases that can be used to manipulate cAMP and cGMP levels in vivo. We identified the blaC gene encoding a putative photoactivated adenylyl cyclase in the Beggiatoa sp. PS genome. BlaC contains a BLUF domain involved in blue-light sensing using FAD and a nucleotidyl cyclase domain. The blaC gene was overexpressed in Escherichia coli, and its product was purified. Irradiation of BlaC in vitro resulted in a small red shift in flavin absorbance, typical of BLUF photoreceptors. BlaC had adenylyl cyclase activity that was negligible in the dark and upregulated by light by 2 orders of magnitude. To convert BlaC into a guanylyl cyclase, we constructed a model of the nucleotidyl cyclase domain and mutagenized several residues predicted to be involved in substrate binding. One triple mutant, designated BlgC, was found to have photoactivated guanylyl cyclase in vitro. Irradiation with blue light of the E. coli cya mutant expressing BlaC or BlgC resulted in the significant increases in cAMP or cGMP synthesis, respectively. BlaC, but not BlgC, restored cAMP-dependent growth of the mutant in the presence of light. Small protein sizes, negligible activities in the dark, high light-to-dark activation ratios, functionality at broad temperature range and physiological pH, as well as utilization of the naturally occurring flavins as chromophores make BlaC and BlgC attractive for optogenetic applications in various animal and microbial models.

Engineering adenylate cyclases regulated by near-infrared window light

“…In this strain, expression of the chromosomal lacZ gene encoding β-galactosidase is low because of the absence of activation by the cAMP-responsive protein (CRP) also known as catabolite activator protein (35). BL21[DE3] cya produces white colonies on agar containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (31). The C-terminal AC domain of CyaB1 (amino acids 585-857) restores lacZ expression, thus generating blue colonies.…”

Algal photoreceptors: in vivo functions and potential applications

“…d Light-regulation of DNA binding activity of the dimeric tryptophan repressor protein TrpR using LOV-Jα; in the dark, binding of LOV to a shared helix of TrpR populates an inactive conformation of the TrpR domain; unfolding of Jα allows for the release of the LOV domain and, thus, activation of the TrpR repressor (modified after Strickland et al 2010). e Utilization of naturally occurring, light-sensitive adenylyl cyclases from the alga Euglena gracilis (euPAC) or the bacterium Beggiatoa (bPAC) to manipulate the cellular cAMP level by light (Ryu et al 2010;Schröder-Lang et al 2007;Stierl et al 2011). f Construction of a light-activated transcription system based on the (rapid) interaction of the cryptochrome CRY2 with CIB1 upon blue light irradiation (modified after Kennedy et al 2010;Liu et al 2012) of neuronal spiking (Boyden et al 2005).…”

LOV and BLUF flavoproteins’ regulatory photoreceptors of microorganisms and photosensory actuators in optogenetic systems

“…У выявленного у бактерий Beggiatoa sp. фото-сенсорного белка, содержащего BLUF-домен, связанный с аденилилциклазой, bPAC (photoactivated adenylyl cyclase) [9] (или BlaC [10]), обнару-жено светоиндуцированное повышение активно-сти этого фермента и уровня клеточного цАМФ (табл. 1).…”

“…Тройной му-тант, BlgC, обладает фотоактивированной гуани-латциклазой in vitro. Синий свет вызывает у мутанта Escherichia coli, экспрессирующего BlaC, значи-тельное повышение уровня цГМФ [10].…”

“…Действительно, светоактивированная bPAC про-являет высокую эффективность в цАМФ-регули-руемых процессах, включая поведенческие реакции, при интеграции c нейронами некоторых животных [9]. Кроме того, BlaC посредством мутации может быть конвертирована в фотоактивируемую цГМФ-циклазу (BlgC), расширяя область ее применения [10]. Как известно, продукты аденилил-и гуани-лилциклаз, цАМФ и цГМФ, являются универсаль-ными вторичными мессенджерами, которые регу-лируют многие процессы у организмов.…”