Computational studies to understand molecular regulation of the TRPC6 calcium channel, the mechanism of purine biosynthesis, and the folding of azobenzene oligomers

Different computational chemistry methods were applied to study a variety of problems at the molecular level. These problems concern protein-protein interactions, the mechanism of reaction for enzymatic purine biosynthesis, structural interconversion in non-natural oligomeric folding, and carbohydrate synthesis.; Transient receptor potential-canonical 6 (TRPC6) calcium channels are currently the subject of intense investigation for their role in modulating smooth muscle tone in blood vessels and lung tissue. Binding of a protein, FKBP12, is a prerequisite for the formation of a multiprotein complex involved in channel regulation. To study the elements of molecular recognition in FKBP12 for binding to the TRPC6 intracellular domain, 20 nanosecond molecular dynamic simulations were performed on the complex of FKBP12 and a peptide model of the wild-type TRPC6 intracellular domain, a phosphorylated Ser768 analog of the wild-type peptide as well as Ser768Asp and Ser768Glu mutants. The phosphorylated peptide demonstrated the greatest binding affinity by the MM-GB/SA method, due to the strong interaction between the phosphate group and two lysine (Lys44 and Lys47) residues of FKBP12 at the binding site. These trajectories also revealed transient, non-simultaneous interactions with the epsilon-NH 3⊕ group of these lysine residues. This feature was not observed in simulations containing the other peptides. Decomposition of the binding free energies into each amino acid residue identified important additional structural elements necessary for this protein-protein interaction.; Potential catalytic reaction mechanisms of the enzyme PurE Class I, which catalyzes the transformation from N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in the purine biosynthetic pathway, were investigated by density functional theory (DFT) methods. The potential energy surfaces (PES) of model processes for these enzymatic reactions have been explored, and have aided in identifying the most energetically feasible pathway. Calculations using a simplified model system, containing only the essential atoms involved in the chemical process, revealed seven potential reaction pathways for transformation of N 5-CAIR to CAIR. The experimental results exclude four of these pathways. Two of the remaining three pathways involve deprotonation of one carbon atom (C4) of the imidazole ring. This process makes the relative energy of transition states of these two pathways higher than the third pathway.; The full N5-CAIR structure was studied via PES calculations, including consideration of the ribose-5-phosphate unit and its different charge states. There are 48 structures of N5-CAIR and CAIR regarding the different protonation states of the substrate. Four reaction pathways were identified based on the available structures after optimization. One pathway involved a cationic substrate. Two involved anionic substrates. A fourth one involved a neutral substrate. These four pathways have similar PES to their counterparts in the simplified model.; The cationic pathway is the most favorable pathway in both the simplified and full reaction models. In this pathway, an intramolecular proton donor/acceptor is required before and after migration of the carboxyl group (-CO2H). According to the spatial arrangement of catalytic amino acids at the active site of the PurE Class I crystal structure (PDB ID: 1D7A), a conserved histidine 45 (His45) residue could be such a proton donor/acceptor. Based on these results, a stepwise enzymatic reaction mechanism for PurE Class I is proposed. First, His45 at the PurE Class I active site protonates the amide nitrogen of N 5-CAIR. Second, the carboxyl group migrates to carbon 4 (C4) with concomitant C-C bond formation. This step generates the protonated CAIR intermediate. In the final step, His45 deprotonates the protonated CAIR intermediate to produce the final product, CAIR, and regenerates it...