Accurate Docking is Achieved by Decoupling Systematic Sampling from Scoring

This dissertation discusses two main projects from my thesis work. The first project focuses on the development of a small molecule docking program, SKATE, for drug discovery. The second project focuses on the critical analysis of the thermal stability of a mini-protein, FSD-1. SKATE is a novel approach to small molecule docking. It removes any inter-dependence between sampling and scoring to improve docking accuracy. SKATE systematically and exhaustively samples a ligand\u27s conformational, rotational and translational degrees of freedom, as constrained by a receptor pocket, to find sterically allowed poses. A total of 266 ligands were re-docked to their respective receptors to assess SKATE\u27s performance. The results show that SKATE was able to sample poses within 2 Angstrom RMSD of the native structure for 97% of the cases. The best performing scoring function was able to rank a pose that is within 2 Angstrom RMSD of the native structure as the top-scoring pose for 83% of the cases. Compared to published data, SKATE has a higher self-docking accuracy rate than or is at least comparable to GOLD, Glide, MolDock and Surflex. The cross-docking accuracy of SKATE was assessed by docking 83 ligands to their respective receptors. The cross-docking results were comparable to those in published methods. Mini-proteins that contain fewer than 50 amino acids often serve as model systems for studying protein folding because their small size makes long time-scale simulations possible. However, not all mini-proteins are created equal. The stability and structure of FSD-1, a 28-residue mini-protein that adopts the Beta Beta Alpha zinc-finger motif independent of zinc binding, was investigated using circular dichroism: CD), differential scanning calorimetry: DSC), and replica-exchange molecular dynamics: REMD). FSD-1\u27s broad melting transition, similar to that of a helix-to-coil transition, was observed in CD, DSC, and REMD experiments. The N-terminal -hairpin was found to be flexible. FSD-1\u27s apparent melting temperature of 41 degrees C may be a reflection of the melting of its alpha-helical segment instead of the entire protein. Thus, FSD-1\u27s status as a model system for studying protein folding should be reconsidered despite its attractiveness for being small in size and it was designed to contain essential helix, sheet, and turn secondary structures. An electronic copy of this dissertation is available online at www.ccb.wustl.edu/~jafen

Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity

AbstractPhosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca2+-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P). Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP. Cosolvents [isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)] also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles. Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates. PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the Tm for PI-PLC thermal denaturation decreased linearly with added iPrOH). However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein. At 30% iPrOH and 4 °C (well below the Tm for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal. iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate. This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little effect on protein secondary structure

Use of Supervisory Control and Data Acquisition for Damage Location of Water Delivery Systems

Urban water delivery systems can be damaged by earthquakes or severely cold weather. In either case, the damage cannot easily be detected and located, especially immediately after the event. In recent years, real-time damage estimation and diagnosis of buried pipelines attracted much attention of researchers focusing on establishing the relationship between damage ratio (breaks per unit length of pipe) and ground motion, taking the soil condition into consideration. Due to the uncertainty and complexity of the parameters that affect the pipe damage mechanism, it is not easy to estimate the degree of physical damage only with a few numbers of parameters. As an alternative, this paper develops a methodology to detect and locate the damage in a water delivery system by monitoring water pressure on-line at some selected positions in the water delivery systems. For the purpose of on-line monitoring, emerging supervisory control and data acquisition technology can be well used. A neural network-based inverse analysis method is constructed for detecting the extent and location of damage based on the variation of water pressure. The neural network is trained by using analytically simulated data from the water delivery system with one location of damage, and validated by using a set of data that have never been used in the training. It is found that the method provides a quick, effective, and practical way in which the damage sustained by a water delivery system can be detected and located

The Assembly of Diverse Immune Receptors Is Focused on a Polar Membrane-Embedded Interaction Site

The majority of receptors responsible for activation of distinct cell types within the immune system assemble with dimeric signaling modules through interaction of a basic transmembrane residue with a pair of acidic residues of the signaling dimer. Because assembly of other membrane proteins requires specific interactions along extended stretches of transmembrane helices, we examined how transmembrane sequences flanking the polar interaction site contribute to assembly for three receptors that associate with different signaling modules—the natural killer cell receptors KIR and NKG2D and the Fc receptor for IgA, FcαRI. The KIR and NKG2D receptors assembled with the DAP12 and DAP10 dimers, respectively, even when the entire KIR or NKG2D transmembrane domains were replaced by polyleucine sequences with a properly positioned basic residue. In contrast, a high degree of specificity for the basic side chain could be observed because the KIR–DAP12 and FcαRI–Fcγ interactions favored lysine or arginine, respectively. Steric hindrance among incompatible extra-membranous domains and competition for signaling modules also contributed to specificity of assembly. These results demonstrate that these interactions are focused on the polar site created by three ionizable transmembrane residues, and explain how the DAP12 and Fcγ signaling modules can assemble with large, non-overlapping sets of receptors that have highly divergent transmembrane sequences