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The work described in this thesis initially focusses on the discovery of a novel PKA-RI specific AKAP: small membrane AKAP (smAKAP). Afterwards we centre on the structural interaction between smAKAP and PKA-RI to reveal the first PKA-RI specific AKAP bound to PKA-RI crystal structure. Interestingly, a novel self-inhibition mechanism was discovered which allows PKA to block its binding to AKAPs under certain restrictions. In order to fully understand the specific limitations associated with binding a study centering on the PKA-RI and PKA-RII interactions with AKAPs was performed. In Chapter 2, recent literature on the structural interface between PKA-RI/RII and AKAPs is reviewed. Most of these structural studies involve either X-ray crystallography, three-dimensional NMR, binding affinity assays and various other biochemical methods. In Chapter 3, the discovery and initial characterization of a novel AKAP termed smAKAP is described. Via binding affinity assays and imaging techniques it is shown that smAKAP is PKA-RI specific. The intracellular location of smAKAP at the plasma membrane is shown by means of fluorescence imaging and advanced electron microscopy. In Chapter 4, structural techniques such as hydrogen/deuterium exchange and X-ray crystallography are applied to probe the interaction interface between smAKAP and PKA-RI. Additionally, via a phosphoproteomics study it was shown that in the middle of the A-kinase binding domain of smAKAP there is a putative PKA phosphorylation site. Upon phosphorylation of this site, PKA cannot bind to smAKAP anymore. A mechanistic model on how this disruption occurs is presented. In Chapter 5, a novel bioinformatic tool, THAHIT (THe AKAP/amphipathic Helix Identification Tool), is able to predict PKA-RIα and/or PKA-RIIα binding domains. This software package is based on currently known and well-established PKA-RIα and PKA-RIIα binding motifs. After applying it on all known AKAPs, numerous new PKA-RIα and PKA-RIIα binding domains in these AKAPs were found and/or narrowed down. Several of these were confirmed via conservation (BlastP), in silico docking studies using HADDOCK and in vitro binding studies using fluorescence anisotropy. In addition, several cAMP pull-downs were investigated for potential novel AKAPs using THAHIT. Here we propose a novel very large AKAP: vlAKAP.

Previously we have shown that the recombinantly produced SA2 amphiphilic oligopeptide (Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu-Glu-COOH) self-assembles into nanovesicles (van Hell et al. 2007). In this study, the intermolecular interactions that contribute to the formation of such peptide vesicles are examined. First, analysis of a 3-hydroxyflavone fluorescent probe inserted into the peptide assemblies demonstrated that the peptide self-assembly is based on hydrophobic clustering. The polarity of this hydrophobic microenvironment was comparable to that of negatively charged lipid bilayers. A substantial level of hydration at the hydrophilic-hydrophobic interface was detected, as was further confirmed by tryptophan fluorescence analysis. However, organic solvents such as acetonitrile, tetrahydrofuran, or ethanol could not disrupt SA2 oligopeptide vesicles, whereas these solvents fully disintegrated lipid vesicles. Instead, the SA2 assembly immediately disintegrated in hydrogen breaking solvents such dimethylsulfoxide and dimethylformamide, suggesting the involvement of additional intermolecular interactions via hydrogen bonding. Circular dichroism and Fourier transform infrared spectroscopy excluded well-defined patterns of intramolecular hydrogen bonding and indicated the polyproline type II as the dominant SA2 peptide conformation, which enables intermolecular hydrogen bonding. All-atom computational simulations were used to confirm the presence of such intermolecular hydrogen bonds and degrees of hydration. On the basis of the experimental and computational data presented, we propose a model of an interdigitated peptide assembly that involves intermolecular hydrogen bonding in addition to hydrophobic interactions that stabilize SA2 oligopeptide vesicles.