Thermodynamics and S-Palmitoylation Dependence of Interactions between Human Aquaporin-4 M1 Tetramers in Model Membranes.

Aquaporin-4 (AQP4) is a water channel protein found primarily in the central nervous system (CNS) that helps to regulate water-ion homeostasis. AQP4 exists in two major isoforms: M1 and M23. While both isoforms have a homotetrameric quaternary structure and are functionally identical when transporting water, the M23 isoform forms large protein aggregates known as orthogonal arrays of particles (OAPs). In contrast, the M1 isoform creates a peripheral layer around the outside of these OAPs, suggesting a thermodynamically stable interaction between the two. Structurally, the M1 isoform has an N-terminal tail that is 22 amino acids longer than the M23 isoform and contains two solvent-accessible cysteines available for S-palmitoylation at cysteine-13 (Cys-13) and cysteine-17 (Cys-17) in the amino acid sequence. Earlier work suggests that the palmitoylation of these cysteines might aid in regulating AQP4 assemblies. This work discusses the thermodynamic driving forces for M1 protein-protein interactions and how the palmitoylation state of M1 affects them. Using temperature-dependent single-particle tracking, the standard state free energies, enthalpies, and entropies were measured for these interactions. Furthermore, we present a binding model based on measured thermodynamics and a structural modeling study. The results of this study demonstrate that the M1 isoform will associate with itself according to the following expressions: 2[AQP4-M1]4 ↔ [[AQP4-M1]4]2 when palmitoylated and 3[AQP4-M1]4 ↔ [AQP4-M1]4 + [[AQP4-M1]4]2 ↔ [[AQP4-M1]4]3 when depalmitoylated. This is primarily due to a conformational change induced by adding the palmitic acid groups at Cys-13 and Cys-17 in the N-terminal tails of the homotetramers. In addition, a statistical mechanical model was developed to estimate the Gibbs free energy, enthalpy, and entropy for forming dimers and trimers. These results were in good agreement with experimental values.

Thermodynamic Driving Forces of Redox-Dependent CPR Insertion into Biomimetic Endoplasmic Reticulum Membranes.

Cytochrome P450 reductase (CPR) is a NADPH-dependent membrane-bound oxidoreductase found in the endoplasmic reticulum (ER) and is the main redox partner for most cytochrome P450 enzymes. Presented are the measured thermodynamic driving forces responsible for how strongly CPR partitions into a biomimetic ER with the same lipid composition of a natural ER. Using temperature-dependent fluorescence correlation spectroscopy and fluorescence single-protein tracking, the standard state free energies, enthalpies, and entropies of the CPR insertion process were all measured. The results of this study demonstrate that the thermodynamic driving forces are dependent on the redox states of CPR. In particular, the partitioning of CPRox into a biomimetic ER is an exothermic process with a small positive change in entropy, while CPRred partitioning is endothermic with a large positive change in entropy. Both resulted in negative free energies and strong association to the biomimetic ER, but the KP of CPRox insertion is measurably smaller than that of CPRred. Using this new information and known results from literature sources, we also present a phenomenological model that accounts for membrane-protein interactions, protein orientation relative to the membrane, and protein conformation as a function of the redox state.