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1.
Photochem Photobiol Sci ; 21(11): 1881-1894, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35984631

RESUMO

Signal transduction typically starts with either ligand binding or cofactor activation, eventually affecting biological activities in the cell. In red light-sensing phytochromes, isomerization of the bilin chromophore results in regulation of the activity of diverse output modules. During this process, several structural elements and chemical events influence signal propagation. In our study, we have studied the full-length bacteriophytochrome from Deinococcus radiodurans as well as a previously generated optogenetic tool where the native histidine kinase output module has been replaced with an adenylate cyclase. We show that the composition of the output module influences the stability of the hairpin extension. The hairpin, often referred as the PHY tongue, is one of the central structural elements for signal transduction. It extends from a distinct domain establishing close contacts with the chromophore binding site. If the coupling between these interactions is disrupted, the dynamic range of the enzymatic regulation is reduced. Our study highlights the complex conformational properties of the hairpin extension as a bidirectional link between the chromophore-binding site and the output module, as well as functional properties of diverse output modules.


Assuntos
Deinococcus , Fitocromo , Conformação Proteica , Cristalografia por Raios X , Fitocromo/química , Sítios de Ligação , Luz , Proteínas de Bactérias/química , Deinococcus/química
2.
Curr Opin Struct Biol ; 57: 72-83, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30878713

RESUMO

Bacteriophytochromes are a subfamily of the diverse light responsive phytochrome photoreceptors. Considering their preferential interaction with biliverdin IXα as endogenous cofactor, they have recently been used for creating optogenetic tools and engineering fluorescent probes. Ideal absorption characteristics for the activation of bacteriophytochrome-based systems in the therapeutic near-infrared window as well the availability of biliverdin in mammalian tissues have resulted in tremendous progress in re-engineering bacteriophytochromes for diverse applications. At the same time, both the structural analysis and the functional characterization of diverse naturally occurring bacteriophytochrome systems have unraveled remarkable differences in signaling mechanisms and have so far only touched the surface of the evolutionary diversity within the family of bacteriophytochromes. This review highlights recent findings and future challenges.


Assuntos
Proteínas de Bactérias/metabolismo , Biologia Celular , Fitocromo/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fitocromo/química , Fitocromo/genética , Engenharia de Proteínas
3.
J Biol Chem ; 293(23): 9078-9089, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29695503

RESUMO

Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light-sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light-regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor-effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen-deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor-effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties.


Assuntos
Adenilil Ciclases/genética , Deinococcus/genética , Guanilato Ciclase/genética , Optogenética/métodos , Fitocromo/genética , Synechocystis/enzimologia , Adenilil Ciclases/química , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Cristalografia por Raios X , Deinococcus/química , Guanilato Ciclase/química , Luz , Simulação de Dinâmica Molecular , Fitocromo/química , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Synechocystis/genética
4.
Sci Adv ; 3(3): e1602498, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28275738

RESUMO

Nature has evolved an astonishingly modular architecture of covalently linked protein domains with diverse functionalities to enable complex cellular networks that are critical for cell survival. The coupling of sensory modules with enzymatic effectors allows direct allosteric regulation of cellular signaling molecules in response to diverse stimuli. We present molecular details of red light-sensing bacteriophytochromes linked to cyclic dimeric guanosine monophosphate-producing diguanylyl cyclases. Elucidation of the first crystal structure of a full-length phytochrome with its enzymatic effector, in combination with the characterization of light-induced changes in conformational dynamics, reveals how allosteric light regulation is fine-tuned by the architecture and composition of the coiled-coil sensor-effector linker and also the central helical spine. We anticipate that consideration of molecular principles of sensor-effector coupling, going beyond the length of the characteristic linker, and the appreciation of dynamically driven allostery will open up new directions for the design of novel red light-regulated optogenetic tools.


Assuntos
Alteromonadaceae/enzimologia , Proteínas de Bactérias/química , Guanilato Ciclase/química , Transdução de Sinais , Regulação Alostérica , Alteromonadaceae/genética , Cristalografia por Raios X , Luz , Domínios Proteicos
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