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Scientific Highlight from ALS Footprinting Program: Local and Global Structural Drivers for the Photoactivation of the Orange Carotenoid Protein.

July 10, 2017

Photoprotective mechanisms are of fundamental importance for the survival of photosynthetic organisms. In cyanobacteria, the orange carotenoid protein (OCP), when activated by intense blue light, binds to the light-harvesting antenna and triggers the dissipation of excess captured light energy. Using a combination of multiple techniques including X-ray footprinting, Gupta et al identified both the local and global structural changes in the OCP upon photoactivation. Microsecond radiolytic labeling identified rearrangement of the H-bonding network associated with conserved residues and structural water molecules. Collectively, these data provide experimental evidence for an ensemble of local and global structural changes, upon activation of the OCP, that are essential for photoprotection.

Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center

June 6, 2017

Carotenoid cleavage oxygenases (CCOs) are non-heme iron enzymes that catalyze scission of alkene groups in carotenoids and stilbenoids to form biologically important products. CCOs possess a rare four-His iron center whose resting-state structure and interaction with substrates are incompletely understood. Here, we address this knowledge gap through a comprehensive structural and spectroscopic study of three phyletically diverse CCOs. The crystal structure of a fungal stilbenoid-cleaving CCO, CAO1, reveals strong similarity between its iron center and those of carotenoid-cleaving CCOs, but with a markedly different substrate-binding cleft. These enzymes all possess a five-coordinate high-spin Fe(II) center withresting-state Fe−His bond lengths of ∼2.15 Å. This ligand set generates an iron environment more electropositive than those of other non-heme iron dioxygenases as observed by Mossbauer isomer shifts. Dioxygen (O2) does not coordinate iron in the absence of substrate. Substrates bind away (∼4.7 Å) from the iron and have little impact on its electronic structure, thus excluding coordination-triggered O2 binding. However, substrate binding does perturb the spectral properties of CCO Fe−NO derivatives, indicating proximate organic substrate and O2-binding sites, which might influence Fe−O2 interactions. Together, these data provide a robust description of the CCO iron center and its interactions with substrates and substrate mimetics that illuminates commonalities as well as subtle and profound structural differences within the CCO family.  

Scientific Highlight from ALS Footprinting Program: Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting

July 1, 2016

The assembly of the Escherichia coli ribosome has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells is only partly understood because assembly is coupled with transcription, modification and processing of the pre-ribosomal RNA. We present a method for footprinting and isolating pre-rRNA as it is synthesized in E. coli cells. Pre-rRNA synthesis is synchronized by starvation, followed by nutrient upshift. RNA synthesized during outgrowth is metabolically labeled to facilitate isolation of recent transcripts. Combining this technique with two in vivo RNA probing methods, hydroxyl radical and DMS footprinting, allows the structure of nascent RNA to be probed over time. Together, these can be used to determine changes in the structures of ribosome assembly intermediates as they fold in vivo.