NMR-Based and Automated Docking Characterization of Protein Structure, Dynamics, and Ligand Binding

NMR-based methods used in conjunction with a technique called docking are used to characterize ligand binding to proteins. Standard NMR methods were used to study the backbone dynamics of substrate binding to phosphomevalonate kinase (PMK) and it was observed that ligand binding caused PMK to undergo large conformational changes. These changes were reflected by the appearance of many chemical shift changes upon binding of the natural substrates of PMK (both the binary and ternary complexes) in 1H-15N HSQC NMR titration experiments. The same process was used to characterize the effect ligand binding has on the many arginines in the active site (and distal to the active site) to determine the effect of long-range coulombic interactions on ligand binding. While studying the backbone dynamics of PMK it was discovered that the N-terminal tail of PMK consisting of 10 residues was very disordered which is unlike every other monphosphate kinase. The function of this N-terminal tail was investigated by attempting to find other proteins in human liver cells that bind this peptide, monitored by ESI mass spectrometry. The thioredoxin system of Mycobacterium Tuberculosis consists of a thioredoxin reductase and three thioredoxins. To help facilitate the understanding of this mechanism the solution structures of the oxidized and the reduced forms of thioredoxin C (TrxC) were solved by NMR and modeled with the crystal structure of the thioredoxin reductase complex. The two redox states of TrxC are very similar to each other with most of the differences coming from subtle changes in the active site of TrxC. Automated docking is the process of computationally determining how a ligand binds to a protein and the correct orientation. A large scale docking study, termed virtual screening, was carried out by docking 10,590 compounds into three proteins to find inhibitors for each protein, and those predicted to bind best were tested experimentally. For each protein there were 3 compounds found to bind with reasonable affinity. When ligands bind to a protein they can undergo dynamic changes. To explore this phenomenon, 15N labeled NAD+ cofactor (and other derivatives) was synthesized and bound to oxidoreductases. Relevant binding motions were monitored using CPMG relaxation NMR experiments

Solution Structures of \u3cem\u3eMycobacterium tuberculosis\u3c/em\u3e Thioredoxin C and Models of Intact Thioredoxin System Suggest New Approaches to Inhibitor and Drug Design

Here, we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C-terminal region. It appears that crystal packing effects have caused an artifactual shift in the α4 helix in the previously reported crystal structure, compared with the solution structure. On the basis of these TrxC structures, chemical shift mapping, a previously reported crystal structure of the M. tuberculosis thioredoxin reductase (not bound to a Trx) and structures for intermediates in the E. coli thioredoxin catalytic cycle, we have modeled the complete M. tuberculosis thioredoxin system for the various steps in the catalytic cycle. These structures and models reveal pockets at the TrxR/TrxC interface in various steps in the catalytic cycle, which can be targeted in the design of uncompetitive inhibitors as potential anti-mycobacterial agents, or as chemical genetic probes of function

A new design concept for indraft wind-tunnel inlets with application to the national full-scale aerodynamic complex

The present inlet design concept for an indraft wind tunnel, which is especially suited to applications for which a specific test section flow quality is required with minimum inlet size, employs a cascade or vaneset to control flow at the inlet plane, so that test section total pressure variation is minimized. Potential flow panel methods, together with empirical pressure loss predictions, are used to predict inlet cascade performance. This concept has been used to develop an alternative inlet design for the 80 x 120-ft wind tunnel at NASA Ames Research Center. Experimental results show that a short length/diameter ratio wind tunnel inlet furnishing atmospheric wind isolation and uniform test section flow can be designed

Health and Financial Fragility: Evidence from Car Crashes and Consumer Bankruptcy

This paper assesses the importance of adverse health shocks as triggers of bankruptcy filings. We view car crashes as a proxy for health shocks and draw on a large sample of police crash reports linked to hospital admission records and bankruptcy case files. We report two findings: (i) there is a strong positive correlation between an individual\u27s pre-shock financial condition and his or her likelihood of suffering a health shock, an example of behavioral consistency; and (ii) after accounting for this simultaneity, we are unable to identify a causal effect of health shocks on bankruptcy filing rates. These findings emphasize the importance of risk heterogeneity in determining financial fragility, raise questions about prior studies of medical bankruptcy, and point to important challenges in identifying the triggers of consumer bankruptcy. JEL Codes: D12, D14, K35

Substrate Induced Structural and Dynamics Changes in Human Phosphomevalonate Iinase and Implications for Mechanism

Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, which is the only pathway for synthesis of isoprenoids and steroids in humans. PMK catalyzes transfer of the γ-phosphate of ATP to mevalonate 5-phosphate (M5P) to form mevalonate 5-diphosphate. Bringing these phosphate groups in proximity to react is especially challenging, given the high negative charge density on the four phosphate groups in the active site. As such, conformational and dynamics changes needed to form the Michaelis complex are of mechanistic interest. Herein, we report the characterization of substrate induced changes (Mg-ADP, M5P, and the ternary complex) in PMK using NMR-based dynamics and chemical shift perturbation measurements. Mg-ADP and M5P Kd\u27s were 6–60 μM in all complexes, consistent with there being little binding synergy. Binding of M5P causes the PMK structure to compress (τc = 13.5 nsec), whereas subsequent binding of Mg-ADP opens the structure up (τc = 15.6 nsec). The overall complex seems to stay very rigid on the psec-nsec timescale with an average NMR order parameter of S2 ∼0.88. Data are consistent with addition of M5P causing movement around a hinge region to permit domain closure, which would bring the M5P domain close to ATP to permit catalysis. Dynamics data identify potential hinge residues as H55 and R93, based on their low order parameters and their location in extended regions that connect the M5P and ATP domains in the PMK homology model. Likewise, D163 may be a hinge residue for the lid region that is homologous to the adenylate kinase lid, covering the “Walker-A” catalytic loop. Binding of ATP or ADP appears to cause similar conformational changes; however, these observations do not indicate an obvious role for γ-phosphate binding interactions. Indeed, the role of γ-phosphate interactions may be more subtle than suggested by ATP/ADP comparisons, because the conservative O to NH substitution in the β-γ bridge of ATP causes a dramatic decrease in affinity and induces few chemical shift perturbations. In terms of positioning of catalytic residues, binding of M5P induces a rigidification of Gly21 (adjacent to the catalytically important Lys22), although exchange broadening in the ternary complex suggests some motion on a slower timescale does still occur. Finally, the first nine residues of the N-terminus are highly disordered, suggesting that they may be part of a cleavable signal or regulatory peptide sequence. Proteins 2009. © 2008 Wiley-Liss, Inc

Depth Camera Aided Dead-Reckoning Localization of Autonomous Mobile Robots in Unstructured GNSS-Denied Environments

In global navigation satellite system (GNSS) denied settings, such as indoor environments, autonomous mobile robots are often limited to dead-reckoning navigation techniques to determine their position, velocity, and attitude (PVA). Localization is typically accomplished by employing an inertial measurement unit (IMU), which, while precise in nature, accumulates errors rapidly, and severely degrades the localization solution. Standard sensor fusion methods, such as Kalman filtering, aim to fuse short-term precise IMU measurements with long-term accurate aiding sensors to establish a precise and accurate solution. In indoor environments, where GNSS and no other a priori information is known about the environment, effective sensor fusion is difficult to achieve, as accurate aiding sensor choices are sparse. Fortunately, an opportunity arises by employing a depth camera in the indoor environment. A depth camera can capture point clouds of the surrounding floors and walls. Extracting attitude from these surfaces can serve as an accurate aiding source, which directly mitigates errors that arise due to gyroscope imperfections. This configuration for sensor fusion leads to a dramatic reduction of PVA error compared to other traditional aiding sensor configurations. This paper provides the theoretical basis for the new aiding sensor method, initial expectations of performance benefit via simulation, and hardware implementation of the new algorithm, thereby verifying its veracity. Hardware implementation is performed on the Quanser Qbot 2™ mobile robot with a Vector-Nav VN-200™ IMU and Kinect™ camera from Microsoft