A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivocyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH₂-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures ∼14 °C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH₂ and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (ΔΔG ≈ 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.

Vertebrate-specific neuronal genes are expected to play a critical role in the diversification and evolution of higher brain functions. Among them, the glycosylphosphatidylinositol (GPI)-anchored netrin-G subfamily members in the UNC6/netrin family are unique in their differential expression patterns in many neuronal circuits, and differential binding ability to their cognate homologous post-synaptic receptors. To gain insight into the roles of these genes in higher brain functions, we performed comprehensive behavioral batteries using netrin-G knockout mice. We found that two netrin-G paralogs that recently diverged in evolution, netrin-G1 and netrin-G2 (gene symbols: Ntng1 and Ntng2, respectively), were responsible for complementary behavioral functions. Netrin-G2, but not netrin-G1, encoded demanding sensorimotor functions. Both paralogs were responsible for complex vertebrate-specific cognitive functions and fine-scale regulation of basic adaptive behaviors conserved between invertebrates and vertebrates, such as spatial reference and working memory, attention, impulsivity and anxiety etc. Remarkably, netrin-G1 and netrin-G2 encoded a genetic “division of labor” in behavioral regulation, selectively mediating different tasks or even different details of the same task. At the cellular level, netrin-G1 and netrin-G2 differentially regulated the sub-synaptic localization of their cognate receptors and differentiated the properties of postsynaptic scaffold proteins in complementary neural pathways. Pre-synaptic netrin-G1 and netrin-G2 diversify the complexity of vertebrate behaviors and differentially regulate post-synaptic properties. Our findings constitute the first genetic analysis of the behavioral and synaptic diversification roles of a vertebrate GPI protein and presynaptic adhesion molecule family.