【Biochem】Computational studies to understand molecular regulation of the TRPC6 calcium channel, the mechanism of purine biosynthesis, and the folding of azobenzene oligomers
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Computational studies to understand molecular regulation of the TRPC6 calcium channel, the mechanism of purine biosynthesis, and the folding of azobenzene oligomers
by Tao, Peng, Ph.D., The Ohio State University, 2007, 499 pages; AAT 3241692
Advisor: Hadad, Christopher M.
School: The Ohio State University
School Location: United States -- Ohio
Index terms(keywords): TRPC6, Calcium channel, Purine, Biosynthesis, Folding, Azobenzene, Oligomers
Source: DAI-B 67/11, May 2007
Source type: Dissertation
Subjects: Chemistry
Publication Number: AAT 3241692
ISBN: 9780542965357
Document URL: [url]http://proquest.umi.com/pqdweb?did=1221738391&sid=19&Fmt=2&RQT=309&VName=PQD[/url]
ProQuest document ID: 1221738391
Abstract (Document Summary)
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 [varepsilon]-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 N 5 -carboxyaminoimidazole ribonucleotide (N 5 -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 N 5 -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 N 5 -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 (-CO 2 H). 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 its initial state. For the first time, an atomistic description of the PurE Class I enzyme catalytic mechanism is provided. This information can be applied to the development of transition state analogs and mechanism-based inhibitors of PurE Class I. These newly designed molecules could be used as potential new drug leads for optimization by synthetic and medicinal chemistry.
Foldamers are defined as unnatural polymers/oligomers with a well-defined, compact, three-dimensional folding capability. Azobenzene units are common linkages in foldamer designs. Four alternating pyridinedicarboxamide/ m -(phenylazo)azobenzene oligomers which could fold into both right- and left-handed helices were studied computationally for their dynamical properties. Two helices were shown as the global minimum among the conformations generated by Monte Carlo simulation. Extended conformations have higher potential energies than compact ones. Molecular dynamics simulations (100 ns) at a single temperature were also performed to study the interconversion process between two helices of these oligomers. However, the molecules were trapped at the local minimum, and no interconversion was observed. To overcome this difficulty, replica-exchange molecular dynamic (REMD) simulations which apply a parallel tempering algorithm were performed on the azobenzene oligomers. Both right- and left-handed helices were successfully sampled in the simulation for all four oligomers. Careful investigation of REMD trajectories revealed twisted conformations as intermediate structures in the interconversion pathway between two helices.
The temperature weighted histogram analysis method (T-WHAM) was applied on the REMD simulation results to generate contour maps of the potential of mean force (PMF). Atomic pair distances and dihedral angles were used as reaction coordinates for PMF contour plots. Analysis showed that right- and left-handed helices are equally sampled in REMD simulations. In large oligomers, both right- and left-handed helices could be adopted by different parts of the molecule simultaneously. The interconversion between two helices could occur in the middle of the helical structure, which is not necessarily at the end of the molecule.
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