Insights into 5-Lipoxygenase Active Site and Catalysis

Leukotrienes are potent immune-modulatory compounds involved in the inflammatory response. 5-Lipoxygenase (5-LOX) is the enzyme that catalyzes the production of Leukotriene A4 (LTA4), which is further metabolized to pro- and anti-inflammatory compounds derived from the substrate Arachidonic Acid (AA), a polyunsaturated fatty acid. The crystal structure of a stabilized form of 5-LOX has been recently reported (Gilbert, Bartlett et al. 2011). However, the structure does not reveal the mode of substrate entry or recognition. The first step in the enzymatic reaction involves the removal of a hydrogen from the central carbon of a pentadiene present in the substrate. It is not known how 5-LOX distinguishes among three such chemically equivalent pentadienes in AA to produce only 5-S-HPETE, which is subsequently transformed to LTA4. The crystal structure suggests a somewhat crescent shaped active site lined with invariant Leucines and Isoleucines, with no clear access port visible for substrate entry. A series of site-directed mutants were produced and the activities of these mutants evaluated. Product analyses of these mutants with AA and substrate analogs suggest entry of the substrate by the opening of the so-called Phenylalanine (F)-Tyrosine(Y) ‘cork’. The data is also consistent with the orientation of AA as carboxyl innermost, as predicted from the stereochemistry of the product and mechanistic models of lipoxygenase function

Ultrafast Time-Resolved Spectroscopy Probes Carboxylate Metal Binding and Proton Transfer Kinetics in Condensed Phase

This dissertation describes two scientific experimental techniques, namely two-dimensional infrared (2D-IR) spectroscopy and time–resolved multiprobe spectroscopy. These methods probe metal coordination geometry in multi-carboxylates and proton transfer kinetics in protic ionic liquids, respectively. Another aspect of this dissertation discusses making the scientific literature accessible to students early in their undergraduate careers. For the metal–carboxylate structural investigation, symmetric stretch vibrational peaks were probed using the EDTA molecule and divalent Ca2+ and Mg2+ ions. Multi–carboxylates are components of proteins. The carboxylate symmetric stretching vibration is sensitive to metal–induced structural changes. In EDTA’s carboxylate stretching region, the symmetric stretch mixes with the CH bending modes. With 2D-IR spectroscopy, density functional theory, participation coefficient analysis, and energy decomposition analysis calculations, the different vibrational contributions were disentangled and associated with the ion binding geometry. Hence, the ion–binding information from EDTA’s symmetric stretch region can be utilized in protein–ion binding research. In the second investigation, time-resolved multiprobe spectroscopy probed a photoacid dissolved in a protic ionic liquid to unravel the proton transfer kinetics in an ionic liquid environment. Our investigation with this system suggests that proton transport has fast kinetics falling within the Grotthuss mechanism and slow rates that match vehicular transport. The time-resolved multiprobe spectroscopy data, ranging from a few hundred femtoseconds to several nanoseconds, reflect the complicated kinetic pathway of the proton in such a system. This work indicates that protic ionic liquids have complex reaction kinetics that must be modeled using the proton conduction models for water. Thirdly, this document describes a curriculum that helps undergraduates assess the research presented in scientific literature. The first semester of this curriculum teaches students to read and comprehend existing original research literature. With worksheet assignments, workshops, and office hours, the students interpret hypotheses, experimental methods, conclusions, and future directions in research. This curriculum provides undergraduates a starting method to evaluate scientific literature and introduces an assignment structure to build on critical thinking skills

Extracting accurate infrared lineshapes from weak vibrational probes at low concentrations

Fourier-transform infrared (FTIR) spectroscopy using vibrational probes is an ideal tool to detect changes in structure and local environments within biological molecules. However, challenges arise when dealing with weak infrared probes, such as thiocyanates, due to their inherent low signal strengths and overlap with solvent bands. In this protocol we demonstrate: • A streamlined approach for the precise extraction of weak infrared absorption lineshapes from a strong solvent background. • A protocol combining a spectral filter, background modeling, and subtraction. • Our methodology successfully extracts the CN stretching mode peak from methyl thiocyanate at remarkably low concentrations (0.25 mM) in water, previously a challenge for FTIR spectroscopy.This approach offers valuable insights and tools for more accurate FTIR measurements using weak vibrational probes. This enhanced precision can potentially enable new approaches to enhance our understanding of protein structure and dynamics in solution

Identification of the Substrate Access Portal of 5-Lipoxygenase

The overproduction of inflammatory lipid mediators derived from arachidonic acid contributes to asthma and cardiovascular diseases, among other pathologies. Consequently, the enzyme that initiates the synthesis of pro-inflammatory leukotrienes, 5-lipoxygenase (5-LOX), is a target for drug design. The crystal structure of 5-LOX revealed a fully encapsulated active site, thus the point of substrate entry is not known. We asked whether a structural motif, a “cork” present in 5-LOX but absent in other mammalian lipoxygenases, might be ejected to allow substrate access. Our results indicate that reduction of cork volume facilitates access to the active site. However, if cork entry into the site is obstructed, enzyme activity is significantly compromised. The results support a model in which the “cork” that shields the active site in the absence of substrate serves as the active site portal, but the “corking” amino acid Phe-177 plays a critical role in providing a fully functional active site. Thus the more appropriate metaphor for this structural motif is a “twist-and-pour” cap. Additional mutagenesis data are consistent with a role for His-600, deep in the elongated cavity, in positioning the substrate for catalysis

Additional file 4: of The impact of biological sex on the response to noise and otoprotective therapies against acoustic injury in mice

OHC loss does not account for the frequency-specific PTS at 16 and 24 kHz or the sex differences in NIHL. Line graph indicating the percentage of OHC loss from apex to base in vehicle-treated noise-exposed animals compared to control non-noise-exposed animals. The frequency range of noise exposure is shaded gray and a gray dotted line outlines the frequency range where significant PTS is seen. Error bars indicate S.E.M. (PDF 396 kb

Additional file 3: of The impact of biological sex on the response to noise and otoprotective therapies against acoustic injury in mice

OHC loss along the cochlear duct. Representative fluorescence microscopy images of the Organ of Corti at the level of the OHC (counter-stained with DAPI) at different frequency bands from controls and mice exposed at 101 dB SPL. There is little to no OHC loss in the control animals, whereas extensive OHC loss is seen above 32 kHz in animals exposed to noise. Scale bar represents 20 μm. (PDF 1120 kb

Additional file 1: of The impact of biological sex on the response to noise and otoprotective therapies against acoustic injury in mice

Schematic showing ABR wave I extraction and analysis. Peak (P1) and trough (T1) values of wave I of the ABR traces (wave shaded in blue) were automatically extracted at stimuli levels from 55 to 85 dB SPL using a MatLab script. Wave I amplitudes were then plotted as a function of the stimuli levels. SigmaPlot was used to perform linear regression (dotted line) and calculate the slope (solid lines). Slopes were then compared between the different groups at 16 kHz. (PDF 471 kb

Additional file 6: of The impact of biological sex on the response to noise and otoprotective therapies against acoustic injury in mice

Effect of noise on pre-synaptic ribbons and active synapses in IHC. Graphs representing the number of pre-synaptic ribbons (a) and active synapses (b) in IHC of control and vehicle-treated noise-exposed animals. A significant decrease in pre-synaptic ribbons and active synapses is observed at 24 and 32 kHz in both males and females, but no difference is seen between sexes. Error bars indicate S.E.M. (PDF 418 kb

Additional file 5: of The impact of biological sex on the response to noise and otoprotective therapies against acoustic injury in mice

Pre-synaptic ribbons and active synapses at 16 kHz and 24 kHz. Representative fluorescence microscopy images of IHC stained for CtBP2 (red) and GluR2 (green) at 16 kHz (left) and 24 kHz (right) from control and noise-exposed mice. The dotted lines represent the approximate border of one IHC. The inset in the bottom left corner image represent a zoom in of active synapses where CtBP2 and GluR2 partially co-localize. Scale bar represents 10 μm. (PDF 1242 kb