Structural Investigation of Binding Events in Proteins

Understanding the biophysical properties that describe protein binding events has allowed for the advancement of drug discovery through structure-based drug design and in silico methodology. The accuracy of these in silico methods depends entirely on the parameters that we determine for them. Many of these parameters are derived from the structural information we have obtained as a community and therein resides the importance of integrity of the quality of this structural data. First, the curation and contents of the Binding MOAD database are extensively described. This database serves as a repository of 25,759 high-quality, ligand-bound X-ray protein crystal structures complemented by 9138 hand-curated binding affinity data for as many of those ligands as appropriate. The newly implemented extended binding site feature is presented, establishing more robust definitions of ligand binding sites than those provided by other databases. Finally, the contents of Binding MOAD are compared to similar databases, establishing the value of our dataset and which purposes it best serves. Second, a robust dataset of 305 unique protein sequences with at least two ligand-bound and two ligand-free structures for each unique protein is cultivated from Binding MOAD and the PDB. Protein flexibility is assessed using C-alpha RMSD for backbone motion and chi-1 angles to quantify side-chain motions. We establish that there is no statistically significant difference between the available conformational space for the backbones or the side chains of unbound proteins when compared to their bound structures. Examining the change in occupied conformational space upon ligand binding reveals a statistically significant increase in backbone conformational space of miniscule magnitude, but a significant increase of side-chain conformational space. To quantify the conformational space available to the side chains, flexibility profiles are established for each amino acid. We found no correlation between backbone and side-chain flexibility. Parallels are then made to common practices in flexible docking techniques. Six binding-site prediction algorithms are then benchmarked on a derivation of the previously established dataset of 305 proteins. We assessed the performance of ligand-bound vs ligand-free structures with these methods and concluded that five of the six methods showed no preference for either structure type. The remaining method, Fpocket, showed decreased performance for ligand-free structures. There was a staggering amount of inconsistency in performance with the methods; different structures of the exact same protein could achieve wildly different rates of success with the same method. The performance of individual structures for all six methods indicated that success and failure rates were seemingly random. Finally, we establish no correlation between the performance of the same structures with different methods, or the performance of the structures with structure resolution, Cruickshank DPI, or number of unresolved residues in their binding sites. Last, we examine the chemical and physical properties of protein-protein interactions (PPIs) with regard to their geometric location in the interface. First, we found that the relative elevation changes of the protein interface landscapes demonstrate that these interfaces are not as flat as previously described. Second, the hollows of druggable PPI interfaces are more sharply shaped and nonpolar in nature, and the protrusions of these druggable PPI interfaces are very polar in character. Last, no correlations exist between the binding affinity describing the subunits of a PPI and other physical and chemical parameters that we measured.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/145943/1/jordanjc_1.pd

Inhibition of type I interferon induction and signalling by mosquito-borne flaviviruses

The Flavivirus genus (Flaviviridae family) contains a number of important human pathogens, including dengue and Zika viruses, which have the potential to cause severe disease. In order to efficiently establish a productive infection in mammalian cells, flaviviruses have developed key strategies to counteract host immune defences, including the type I interferon response. They employ different mechanisms to control interferon signal transduction and effector pathways, and key research generated over the past couple of decades has uncovered new insights into their abilities to actively decrease interferon antiviral activity. Given the lack of antivirals or prophylactic treatments for many flaviviral infections, it is important to fully understand how these viruses affect cellular processes to influence pathogenesis and disease outcome. This review will discuss the strategies mosquito-borne flaviviruses have evolved to antagonise type I interferon mediated immune responses

ROLE OF CYCLOPHILIN D IN SECONDARY SPINAL CORD AND BRAIN INJURY

In the hours and days following acute CNS injury, a secondary wave of events is initiated that exacerbate spinal tissue damage and neuronal cell death. A potential mechanism driving these secondary events is opening of the mitochondrial permeability transition pore (mPTP) and subsequent release of several cell death proteins. Previous studies have shown that inhibition of cyclophilin D(CypD), the key regulating component in mPTP opening, was protective against insults that induce necrotic cell death. We therefore hypothesized that CypD-null mice would show improved functional and pathological outcomes following spinal cord injury (SCI) and traumatic brain injury (TBI). Moderate and severe spinal contusion was produced in wild-type (WT) and CypD-null mice at the T-10 level using the Infinite Horizon impactor. Changes in locomotor function were evaluated using the Basso Mouse Scale (BMS) at 3 days post-injury followed by weekly testing for 4 weeks. Histological assessment of tissue sparing and lesion volume was performed 4 weeks post SCI. Calpain activity, measured by calpain-mediated spectrin degradation, was assessed in moderate injury only by western blot 24 hours post SCI. Results showed that following moderate SCI, CypD-null mice had no significant improvement in locomotor recovery or tissue sparing compared to wild-type mice. Following severe SCI, CypD-null mice showed significantly lower locomotor recovery and decreased tissue sparing compared to WT mice. Calpain-mediated spectrin degradation was not significantly reduced in CypD-null mice compared to WT mice 24h post moderate SCI. The lack of protective effects in CypD-null mice suggests that more dominant mechanisms are involved in the pathology of SCI. In addition, CypD may have a pro survival role that is dependent on the severity of the spinal cord injury

The Power Of A Flying Pig: Carl Stalling, Looney Tunes, And America\u27s Need For Escape.

This thesis examines the creation of Looney Tunes cartoons and how the powerful pairing of music and animation created a convincing reality and escape for audiences. Looney Tunes was created in 1933 by Warner Brothers animation under the stipulation that one song from a Warner Brothers feature film was used in each short. In 1936 the company hired composer Carl Stalling, who worked closely with the animation directors, progressively learning how to better pair music and animation. Stalling often used familiar popular, classical, and folk melodies within his compositions. An analysis of the Looney Tunes short “Speaking of the Weather” (1937), shows how Stalling wove these different genres of music together. An analysis of the short “There They Go-Go-Go” (1956), shows how Stalling’s composition style and coordination with animation developed. This analysis demonstrates how Stalling’s compositions are built out of repeated memorable motives, coordinated with specific actions in the animation. Drawing on theories from film and media psychology, these analyses demonstrate how Looney Tunes created a psychological escape for audiences

Partially Closed Valve Effects on Electromagnetic Flow Meter Accuracy

As the need to manage water resources increases, the ability to accurately measure use becomes crucial. In industry, measurement inaccuracies can cost either the supplier or consumer large amounts of money or even result in a depleted resource. Generally, flow meters need a flow profile to be fully developed to optimize measurement accuracy. In order to produce accurate measurements, most flow meters require a straight section of pipe immediately upstream of the meter. The straight pipe helps to develop the flow profile and produces flow conditions that are as ideal as possible. In many field installations, space restrictions lead to meters being installed with reduced straight pipe between a flow disturbance and the meter. Electromagnetic flow meters are referred to by several different names including: magnetic flow meter, mag meter or mag. The electromagnetic flow meter has grown in popularity in recent years as technology has improved and costs have decreased. Manufacturers commonly claim that their meters can produce highly accurate flow measurements, some as low as ±0.10%, with reduced upstream straight pipe requirements as low as 0 pipe diameters between a disturbance and the meter itself. The results of this study provide quantified error associated with a partially closed butterfly valve on electromagnetic flow meter accuracy. The results show the effect of four valve openings (25%, 50%, 75%, and 100% open), at five locations downstream of the valve (1, 3, 5, 10, and 20 pipe diameters), and at two different meter orientations to the valve shaft. It was concluded in the study that twenty pipe diameters of length are required to produce accurate measurements (within the manufacturer’s specified accuracy) when a magnetic flow meter is installed downstream of a throttling butterfly valve. Surprisingly, the largest effect to the meter’s accuracy unexpectedly occurred when the upstream butterfly valve was fully open and not when it was in a throttling position

Reviews

Danny Saunders and Nina Smalley (eds.), The International Simulation and Gaming Research Yearbook — Volume 8: Simulations and Games for Transition and Change, London: Kogan Page, 2000. ISBN: 0–7494–3397–3. Hardback, viii+271 pages, £40.00