The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy
Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases, and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key...
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eLife Sciences Publications Ltd
2021
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oai:doaj.org-article:9c4cde73b709437591d114b29a509df22021-11-08T17:07:36ZThe inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy10.7554/eLife.713842050-084Xe71384https://doaj.org/article/9c4cde73b709437591d114b29a509df22021-10-01T00:00:00Zhttps://elifesciences.org/articles/71384https://doaj.org/toc/2050-084XEnzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases, and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here, we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions.Paul FischerShatanik MukherjeeEnrico PeterMatthias BroserFranz BartlPeter HegemanneLife Sciences Publications LtdarticleFTIRenzymerhodopsincaged compoundcGMPrhodopsin guanylyl cyclasecatenaria anguillulaeMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021) |
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FTIR enzymerhodopsin caged compound cGMP rhodopsin guanylyl cyclase catenaria anguillulae Medicine R Science Q Biology (General) QH301-705.5 |
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FTIR enzymerhodopsin caged compound cGMP rhodopsin guanylyl cyclase catenaria anguillulae Medicine R Science Q Biology (General) QH301-705.5 Paul Fischer Shatanik Mukherjee Enrico Peter Matthias Broser Franz Bartl Peter Hegemann The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy |
| description |
Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases, and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here, we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions. |
| format |
article |
| author |
Paul Fischer Shatanik Mukherjee Enrico Peter Matthias Broser Franz Bartl Peter Hegemann |
| author_facet |
Paul Fischer Shatanik Mukherjee Enrico Peter Matthias Broser Franz Bartl Peter Hegemann |
| author_sort |
Paul Fischer |
| title |
The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy |
| title_short |
The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy |
| title_full |
The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy |
| title_fullStr |
The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy |
| title_full_unstemmed |
The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy |
| title_sort |
inner mechanics of rhodopsin guanylyl cyclase during cgmp-formation revealed by real-time ftir spectroscopy |
| publisher |
eLife Sciences Publications Ltd |
| publishDate |
2021 |
| url |
https://doaj.org/article/9c4cde73b709437591d114b29a509df2 |
| work_keys_str_mv |
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