Event Title

Crystal Structures Of A Phytochrome From The Non-Photosynthetic Myxobacterium S. Aurantiaca

Location

Lobby in front of Auditorium

Start Date

19-4-2019 11:00 AM

Department

Biology

Description

Bacteriophytochromes (BphPs) are red light photoreceptors found in photosynthetic and non- photosynthetic bacteria. They are dimeric proteins that consist of a photo-sensory core module (PCM) and an enzymatic domain, which are often histidine kinases. The PCM consists of the Per/Arnt/Sim (PAS) domain, the guanosine monophosphate phosphodiesterase / adenylyl cyclase / FhlA (GAF) domain, and a phytochrome-specific (PHY) domain. The PHY domain is covalently linked to an effector domain with histidine kinase activity. The GAF domain contains a covalently bound chromophore called biliverdin (BV) which is a heme-derived, open chain tetrapyrrole. The BphPs reversibly interconverts between the red-light absorbing Pr state and the far-red light absorbing Pfr state. The non-photosynthetic myxobacterium Stigmatella aurantiaca contains two bacteriophytochromes (BphPs) denoted SaBphP1 and SaBphP2. Interestingly, SaBphP1 lacks a conserved histidine in the GAF domain, which stabilizes the Pr state by forming a hydrogen bond with the D-ring of BV. The same histidine also stabilizes the Pfr state by forming a hydrogen bond with the C-ring propionate side chain. SaBphP2, like classical phytochromes, contains the mentioned conserved histidine and shares the same domain composition as SaBphP1. Recently, we determined two crystal structures of SaBphP2 PCM: a) in the wild-type and b) in a mutant form that contains threonine instead of conserved histidine in the GAF domain. These two variants of SaBphP2 PCM crystallize in the same space group (P2 1 ) and diffract to 1.9 and 2.2 Ångstrom resolution, respectively. When compared, the structural differences between two proteins are observed at the dimer interface and in the chromophore-binding pocket, explaining the lack of photoactivity in the SaBphP2 mutant. Through our recent developments of highly-diffracting SaBphP2 microcrystals, we propose SaBphP2 to be a suitable model for investigations at X-ray Free Electron Lasers (XFELs) using Time-Resolved Serial Femtosecond X-ray crystallography (TR-SFX). Recently, the potential of X-FELs for tracking the dynamics of light-triggered processes such as the ones observed in BphPs by TR-SFX has been demonstrated using micron-sized crystals. TR-SFX is a method in which micro-crystals supplied via injector or fixed target delivery systems are exposed to the X-rays at room temperature to obtain important structural intermediates of light-triggered reactions. The proposed experiments will provide insight in the progress of the photo-reaction and in the formation of intermediates during of the Pr to Pfr transition. This will then help to determine how enzymatic activity is controlled and regulated by the light stimulus in phytochromes in general. Funded by NSF MCB RUI 1413360 and NSF MCB EAGER 1839513 Research Grants to E.A.S. and NSF STC “BioXFEL” (1231306).

Comments

Emina Stojković and Marius Schmidt are the faculty sponsors of this poster.

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Apr 19th, 11:00 AM

Crystal Structures Of A Phytochrome From The Non-Photosynthetic Myxobacterium S. Aurantiaca

Lobby in front of Auditorium

Bacteriophytochromes (BphPs) are red light photoreceptors found in photosynthetic and non- photosynthetic bacteria. They are dimeric proteins that consist of a photo-sensory core module (PCM) and an enzymatic domain, which are often histidine kinases. The PCM consists of the Per/Arnt/Sim (PAS) domain, the guanosine monophosphate phosphodiesterase / adenylyl cyclase / FhlA (GAF) domain, and a phytochrome-specific (PHY) domain. The PHY domain is covalently linked to an effector domain with histidine kinase activity. The GAF domain contains a covalently bound chromophore called biliverdin (BV) which is a heme-derived, open chain tetrapyrrole. The BphPs reversibly interconverts between the red-light absorbing Pr state and the far-red light absorbing Pfr state. The non-photosynthetic myxobacterium Stigmatella aurantiaca contains two bacteriophytochromes (BphPs) denoted SaBphP1 and SaBphP2. Interestingly, SaBphP1 lacks a conserved histidine in the GAF domain, which stabilizes the Pr state by forming a hydrogen bond with the D-ring of BV. The same histidine also stabilizes the Pfr state by forming a hydrogen bond with the C-ring propionate side chain. SaBphP2, like classical phytochromes, contains the mentioned conserved histidine and shares the same domain composition as SaBphP1. Recently, we determined two crystal structures of SaBphP2 PCM: a) in the wild-type and b) in a mutant form that contains threonine instead of conserved histidine in the GAF domain. These two variants of SaBphP2 PCM crystallize in the same space group (P2 1 ) and diffract to 1.9 and 2.2 Ångstrom resolution, respectively. When compared, the structural differences between two proteins are observed at the dimer interface and in the chromophore-binding pocket, explaining the lack of photoactivity in the SaBphP2 mutant. Through our recent developments of highly-diffracting SaBphP2 microcrystals, we propose SaBphP2 to be a suitable model for investigations at X-ray Free Electron Lasers (XFELs) using Time-Resolved Serial Femtosecond X-ray crystallography (TR-SFX). Recently, the potential of X-FELs for tracking the dynamics of light-triggered processes such as the ones observed in BphPs by TR-SFX has been demonstrated using micron-sized crystals. TR-SFX is a method in which micro-crystals supplied via injector or fixed target delivery systems are exposed to the X-rays at room temperature to obtain important structural intermediates of light-triggered reactions. The proposed experiments will provide insight in the progress of the photo-reaction and in the formation of intermediates during of the Pr to Pfr transition. This will then help to determine how enzymatic activity is controlled and regulated by the light stimulus in phytochromes in general. Funded by NSF MCB RUI 1413360 and NSF MCB EAGER 1839513 Research Grants to E.A.S. and NSF STC “BioXFEL” (1231306).