cGMP-dependent protein kinases (PKG) are key players in intracellular second messenger signaling within many cellular systems throughout the body. Most notably PKG is known for its role in smooth muscle relaxation (Pfeiffer et.al, 1999). The Iα PKG isozyme has been identified as the primary effector of the nitric oxide pathway (and serves to be a novel drug target). To date the overall knowledge of structure and function of PKG is lacking in terms of the mechanisms of activation and the structural orientations that coordinate them. Recently, our laboratory has solved the crystal structure of the regulatory domain of PKG Iα, which revealed a previously unknown α-helical domain dubbed the Switch Helix (SW) (Osborne et.al, 2011). The SW domain was found to be a site of interprotomer communication via hydrophobic interactions between its C-terminus and hydrophobic residues, named the nest located on the opposing protomer. Synthetic peptides derived from the SW domain, named S-tides, dosedependently activate PKG Iα (Moon et.al, 2015). In addition, the amino acid residues of the nest are in proximity to the cGMP binding site B. It was hypothesized that the binding site for S-tides (nest) and the cGMP binding site B interact and are co-dependent of one another. The hypothesis of this thesis is the binding site for the S-tides (nest) and the cGMP binding site B interact and are co-dependent of one another. To test this hypothesis two aims were constructed: Aim 1: To develop an S1.5 analog that utilizes both the nest and the B-site to increase S-tide activity, Aim 2: To explore the intricacies of these modes of activation and how they interact with each other to obtain a better understanding of the interplay between these two sites. First, based on the most potent S-tide S1.5 (YEDAEAKAKYEAEAAFFANLKLSD, Ka=6 μM), two analogs were synthesized. The peptide S2.5 which lacked the amino acids LSD at the C-terminus showed a three-fold lower activation constant (Ka= 15 μM), although the molecule retained its helicity as demonstrated by circular dichroism. The second analog, S3.5 contained unnatural amino acid components from a molecular modeling approach in an effort to further increase the affinity by interacting with the adjacent cGMP binding site B. However, S3.5 showed further reduction in activity with an activation constant of 70 μM. These findings led us to conclude that the failure of the SAR approach indicates a different mode of S-tide activation as had been previously thought. Next, we investigated the role of the cGMP binding site B in the mechanism of S-tide mediated PKG Iα activation. Co-activation assays with cGMP and S1.5 demonstrated that cGMP activation is not altered in the presence of S1.5. Furthermore, S1.5 mediated activation is negatively affected in the presence of cGMP. These results suggest that the B-site of cGMP does not positively enforce the S1.5 activation kinetics. Next, we employed the PKG Iα mutant E292A, which cannot bind cGMP to the B-site (Moon et.al., 2018). Interestingly, this mutant retains the activation kinetics of PKG Iα WT when activated via S1.5 and cGMP. Thus, the cGMP binding site B is not crucial in the activation mechanisms of activating PKG Iα with cGMP. Likewise, the cGMP binding site B is not crucial in the activation mechanisms of activating PKG Iα with S1.5. To further support these findings, the PKG Iα mutant C42A, which showed crippled cGMP activation kinetics could be activated with S1.5 with a potency similar to wild type. Taken together, the results in this thesis demonstrate that in contrast to the initial hypothesis the binding sites for S-tides and cGMP, although in proximity, show no experimental support of a positive interaction. These findings are significant as they reveal that S1.5 mediated activation of PKG is truly independent of cGMP, thereby providing a molecular platform for the therapeutic development of these unique peptides.