Characterization and engineering of photoactivated adenylyl cyclases

Abstract

Cyclic nucleoside monophosphates (cNMP) serve as universal second messengers in signal transduction across prokaryotes and eukaryotes. As signaling often relies on transiently formed microdomains of elevated second messenger concentration, means to precisely perturb the spatiotemporal dynamics of cNMPs are uniquely poised for the interrogation of the underlying physiological processes. Optogenetics appears particularly suited as it affords light-dependent, accurate control in time and space of diverse cellular processes. Several sensory photoreceptors function as photoactivated adenylyl cyclases (PAC) and hence serve as light-regulated actuators for the control of intracellular levels of 3′, 5′-cyclic adenosine monophosphate. To characterize PACs and to refine their properties, we devised a test bed for the facile analysis of these photoreceptors. Cyclase activity is monitored in bacterial cells via expression of a fluorescent reporter, and programmable illumination allows the rapid exploration of multiple lighting regimes. We thus probed two PACs responding to blue and red light, respectively, and observed significant dark activity for both. We next engineered derivatives of the red-light-sensitive PAC with altered responses to light, with one variant, denoted DdPAC, showing enhanced response to light. These PAC variants stand to enrich the optogenetic toolkit and thus facilitate the detailed analysis of cNMP metabolism and signaling.

PACmn for improved optogenetic control of intracellular cAMP

“…Albeit low, the dark activity of bPAC(wt) 0.057 min -1 (0.0009 s -1 ) is sufficient to increase resting cAMP in the absence of light, in agreement with other reports [44,52,53]. In consequence, about 25% of Drosophila larvae display nocifensive-like bending behavior in the absence of Fig.…”

Signal transduction in photoreceptor histidine kinases

“…Where present in the structure, the effectors are mostly connected to the PCM via a continuous parallel coiled coil, thus possibly restricting the quaternary structures the PCM can adopt in the Pr and Pfr states. In particular, it remains to be seen whether the large‐scale splaying apart of the PHY domains observed for the isolated Dr BPhy PCM manifests to the same extent within the context of full‐length native and engineered BPhy receptors . In fact, recent structures of a BPhy‐GGDEF enzyme indicate that the conformational transitions within the context of a full‐length receptor are of much smaller amplitude .…”

“…In particular, it remains to be seen whether the large-scale splaying apart of the PHY domains observed for the isolated DrBPhy PCM manifests to the same extent within the context of fulllength native and engineered BPhy receptors. 105,[111][112][113][114][115] In fact, recent structures of a BPhy-GGDEF enzyme indicate that the conformational transitions within the context of a fulllength receptor are of much smaller amplitude. 107 Regardless, the long continuous α helices are conducive to downstream relay of the conformational signal, see below.…”

Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered

“…Even prior to the present elucidation of the enzymatic activity of DrBphP, its PSM has provided a versatile building block for novel light-controllable enzymes to be used in optogenetics. Pertinent enzymes have for instance been constructed through fusion of the DrBphP PSM with a cyclicmononucleotide phosphodiesterase 56,57 , a guanylate/adenylate cyclase 58,59 , and a tyrosine kinase 60 . Our study introduces a red light-regulated HK chimera and phosphatase as a potential addition to the optogenetic toolkit.…”