The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy

eLife

Paul Fischer¹  Peter Hegemann¹  Enrico Peter¹  Matthias Broser¹  Franz Bartl²  Shatanik Mukherjee² 

[1] Institute for Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany;Institute of Biology, Biophysical Chemistry, Humboldt University of Berlin, Berlin, Germany;

关键词: FTIR;    enzymerhodopsin;    caged compound;    cGMP;    rhodopsin guanylyl cyclase;    catenaria anguillulae;    optogenetic;    Other;   

DOI  :  10.7554/eLife.71384

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY