Methods to investigate structure and activation dynamics of GC-1/GC-2

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme consisting of one α and one β subunit. The α₁β₁ (GC-1) and α₂β₁ (GC-2) heterodimers are important for NO signaling in humans and catalyse the conversion from GTP to cGMP. Each sGC subunit consists of four domains. Several crystal structures of the isolated domains are available. However, crystals of full-length sGC have failed to materialise. In consequence, the detailed three dimensional structure of sGC remains unknown to date. Different techniques including stopped-flow spectroscopy, Förster-resonance energy transfer, direct fluorescence, analytical ultracentrifugation, chemical cross-linking, small-angle X-ray scattering, electron microscopy, hydrogen-deuterium exchange and protein thermal shift assays, were used to collect indirect information. Taken together, this circumstantial evidence from different groups brings forth a plausible model of sGC domain arrangement, spatial orientation and dynamic rearrangement upon activation. For analysis of the active conformation the stable binding mode of sGC activators has a significant methodological advantage over the transient, elusive, complex and highly concentration dependent effects of NO in many applications. The methods used and the results obtained are reviewed and discussed in this article.