The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability

Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine, the first step in the synthesis of catecholamine neurotransmitters. The protein contains a 159-residue regulatory domain (RD) at its N-terminus that forms dimers in solution; the N-terminal region of RDTyrH (residues 1–71) is absent in the solution structure of the domain. We have characterized the conformational stability of two species of RDTyrH (one containing the N-terminal region and another lacking the first 64 residues) to clarify how that N-terminal region modulates the conformational stability of RD. Under the conditions used in this study, the RD species lacking the first 64 residues is a monomer at pH 7.0, with a small conformational stability at 25 °C (4.7 ± 0.8 kcal mol^–1). On the other hand, the entire RDTyrH is dimeric at physiological pH, with an estimated dissociation constant of 1.6 μM, as determined by zonal gel filtration chromatography; dimer dissociation was spectroscopically silent to circular dichroism but not to fluoresecence. Both RD species were disordered below physiological pH, but the acquisition of secondary native-like structure occurs at pHs lower than those measured for the attainment of tertiary native- and compactness-like arrangements

Mutation of Ser-50 and Cys-66 in Snapin Modulates Protein Structure and Stability

Snapin is a 15 kDa protein present in neuronal and non-neuronal cells that has been implicated in the regulation of exocytosis and endocytosis. Protein kinase A (PKA) phosphorylates Snapin at Ser-50, modulating its function. Likewise, mutation of Cys-66, which mediates protein dimerization, impairs its cellular activity. Here, we have investigated the impact of mutating these two positions on protein oligomerization, structure, and thermal stability, along with the interaction with SNARE proteins. We found that recombinant purified Snapin in solution appears mainly as dimers in equilibrium with tetramers. The protein exhibits modest secondary structure elements and notable thermal stability. Mutation of Cys-66 to Ser abolished subunit dimerization, but not higher-order oligomers. This mutant augmented the presence of α-helical structure and slightly increased the protein thermal stability. Similarly, the S50A mutant, mimicking the unphosphorylated protein, also exhibited a higher helical secondary structure content than the wild type, along with greater thermal stability. In contrast, replacement of Ser-50 with Asp (S50D), emulating the protein-phosphorylated state, produced a loss of α-helical structure, concomitant with a decrease in protein thermal stability. In vitro, the wild type and mutants weakly interacted with SNAP-25 and the reconstituted SNARE complex, although S50D exhibited the strongest binding to the SNARE complex, consistent with the observed higher cellular activity of PKA-phosphorylated Snapin. Our observations suggest that the stronger binding of S50D to SNAREs might be due to a destabilization of tetrameric assemblies of Snapin that favor the interaction of protein dimers with the SNARE proteins. Therefore, phosphorylation of Ser-50 has an important impact on the protein structure and stability that appears to underlie its functional modulation