DOI: 10.5061/dryad.6wwpzgmzx
title: 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.
description: <strong>Preparation</strong> To prepare samples for FTIR, 500 µl of the initial protein solution (1 OD) was concentrated with an Amicon Ultra 10 kDa centrifugal filter to a final OD of ~33 at 540 nm. 10-15 µl of this solution was then placed on a BaF<sub>2</sub> window and concentrated by evaporating solvent water under a stream of dry air. For the CaRGC protein, this step has to be performed with much care to prevent complete dehydration of the sample, since it induces irreversible denaturation of the protein. Samples were then sealed with a second BaF<sub>2</sub> window. To ensure reproducible and constant sample thickness, a 6 µm PTFE spacer was placed between the windows. For measurements between 900 and 1800 cm<sup>−1</sup>, an optical cutoff filter at 1850 cm<sup>−1</sup> was placed in the beamline. The spectral resolution was 2 cm<sup>−1</sup>. Illumination was performed with a pulsed laser for uncaging experiments (330 nm, 6 ns, 10 Hz, 30 mJ per pulse) and time resolved measurements (532 nm). While for continuous illumination a 50 mW continuous-wave (CW), a laser with an output maximum of 532 nm was used (no. 37028, Edmund Optics, York, UK). LED illumination was performed with a set of 520 nm LEDs with a FWHM&gt;20 nm.  Acquired data was initially processed using OPUS 7.5 software, whereas further processing, including baseline correction with a linear function and pre-spline as well as SVD and global fit procedures, was performed by a customized software developed for Octave 5.1.0.0 initially conceived by Dr. Eglof Ritter. <b>Enzyme turnover</b> All samples were prepared under red light &gt;640 nm. Caged compounds NPE-GTP and NPE-ATP were purchased from Jena Bioscience GmbH (Jena, Germany). For reference, measurements on GTP, cGMP and PP, as well as the caged ATP and GTP compounds the substrate to Mn<sup>2+</sup> ratio was 1:2. For measurements of the catalytic activity, the caged compound (NPE-GTP/ATP 50 µl, 10 mM) and manganese (MnCl<sub>2</sub>·4H<sub>2</sub>O 10 µl, 100 mM) were added to the diluted protein solution (1 OD) to ensure sufficient diffusion. After an incubation period of 30 min in the dark, the sample was concentrated with an Amicon Ultra 10 kDa centrifugal filter as described by the manufacturer. <b>RGC homology model</b> The CaRGC-43 model was generated using CHARMM and PyMol 2.5 based on a homology model of its rhodopsin and linker domains. The crystal structures of the rhodopsin phosphodiesterase from Salpingoeca rosetta SrRhoPDE (PDB-IDs: 7CJ3, 7D7Q)<sup>34</sup> served as templates for homology modelling using the online platforms for protein structure prediction of Swiss-Modell and Robetta. The crystal waters and the orientation of both protomers were adopted from the template structures. Secondary structure prediction on the full-length CaRGC sequence in JPred4 helped to identify several additional N- and C-terminal features besides the 7-TM-rhodopsin or GC domains as the following: an additional TM-helix 0, an elongated TM-helix 7, short helices on both the N-terminus (helix -1) and the C-terminus (helix 8) and short N-terminal β-sheets. These structures were modelled using CHARMM and then oriented and linked in PyMol in which the cryo-EM maps of the NO-activated human soluble guanylate cyclase (sGC) served as a template (EMDB-ID: EMD-9885). The final 43-truncated rhodopsin domain was linked to the crystal structure of the guanylate cyclase domain of RhGC in complex with GTP (PDB-ID: 6SIR) using PyMol.
description: This dataset contains the depicted absorbance and difference
 absorbance spectra and homology model *.pdb-files presented in the
 manuscript entitled "The inner mechanics of Rhodopsin Guanylyl
 Cyclase during cGMP-formation revealed by real-time FTIR
 spectroscopy". Each spectrum is labeled according to the manuscript
 figure with a unique identifier and consists of two columns which
 represent the wavenumber and absorbance or difference absorbance
 information sepratated by a comma sign. The spectra can be plotted by any
 commonly used plotting program (e.g. Octave, GNU, Origin, etc.). The
 homology models depicted in the manuscript appendix are labeled
 accordingly and can be displayed and manipulated with the
 open-source molecular visualization system PyMol
 (v.2.4.0).