Cyclic nucleotide-gated (CNG) ion channels are vital for cellular signaling and excitability, with activation regulated by cyclic adenosine- or guanosine-monophosphate (cAMP, cGMP) binding. However, the allosteric mechanisms underlying this activation, particularly the energetics that describes conformational changes within individual domains and between domains, remain unclear. The prokaryotic CNG channel SthK has been a useful model for better understanding these allosteric mechanisms. Here, we applied genetic code expansion (GCE) and time-resolved transition metal ion Förster resonance energy transfer (tmFRET) to investigate the conformational dynamics and energetics in the cyclic nucleotide-binding domain (CNBD) of SthK in both a soluble C-terminal fragment of the protein, SthK Cterm , and in the full-length channel, SthK Full . We incorporated the noncanonical amino acid Acd as a FRET donor and a metal bound to a chelator conjugated to a cysteine as an acceptor. We used time-correlated single-photon counting (TCSPC) to measure time-resolved FRET and fit the TCSPC data to obtain donor-acceptor distance distributions in the absence and presence of cAMP. The distance distributions allowed us to quantify the energetics of coupling between the C-terminal domains and the transmembrane domains by comparing the donor-acceptor distance distributions for SthK Cterm and SthK Full . Our data indicate that the presence of the SthK transmembrane domains makes the activating conformational change in the CNBD more favorable. These findings highlight the power of GCE and time-resolved tmFRET to uncover the structural and energetic landscapes of allosteric proteins and of the ligand-mediated mechanism in CNG channels specifically.
Eggan et al. (Sun,) studied this question.