Project description:The solution structure of human adult carbonmonoxy hemoglobin (HbCO A) was refined using stereospecifically assigned methyl groups and residual dipolar couplings based on our previous nuclear magnetic resonance structure. The tertiary structures of individual chains were found to be very similar to the X-ray structures, while the quaternary structures in solution at low salt concentrations resembled the X-ray R structure more than the R2 structure. On the basis of chemical shift perturbation by inositol hexaphosphate (IHP) titration and docking, we identified five possible IHP binding sites in HbCO A. Amide-water proton exchange experiments demonstrated that αThr38 located in the α1β2 interface and several loop regions in both α- and β-chains were dynamic on the subsecond time scale. Side chain methyl dynamics revealed that methyl groups in the α1β2 interface were dynamic, but those in the α1β1 interface were quite rigid on the nanosecond to picosecond and millisecond to microsecond time scales. All the data strongly suggest a dynamic α1β2 interface that allows conformational changes among different forms (like T, R, and R2) easily in solution. Binding of IHP to HbCO A induced small structural and dynamic changes in the α1β2 interface and the regions around the hemes but did not increase the conformational entropy of HbCO A. The binding also caused conformational changes on the millisecond time scale, very likely arising from the relative motion of the α1β1 dimer with respect to the α2β2 dimer. Heterotropic effectors like IHP may change the oxygen affinity of Hb through modulating the relative motion of the two dimers and then further altering the structure of heme binding regions.
Project description:Cationic antimicrobial peptides and their therapeutic potential have garnered growing interest because of the proliferation of bacterial resistance. However, the discovery of new antimicrobial peptides from animals has proven challenging due to the limitations associated with conventional biochemical purification and difficulties in predicting active peptides from genomic sequences, if known. As an example, no antimicrobial peptides have been identified from the American alligator, Alligator mississippiensis, although their serum is antimicrobial. We have developed a novel approach for the discovery of new antimicrobial peptides from these animals, one that capitalizes on their fundamental and conserved physico-chemical properties. This sample-agnostic process employs custom-made functionalized hydrogel microparticles to harvest cationic peptides from biological samples, followed by de novo sequencing of captured peptides, eliminating the need to isolate individual peptides. After evaluation of the peptide sequences using a combination of rational and web-based bioinformatic analyses, forty-five potential antimicrobial peptides were identified, and eight of these peptides were selected to be chemically synthesized and evaluated. The successful identification of multiple novel peptides, exhibiting antibacterial properties, from Alligator mississippiensis plasma demonstrates the potential of this innovative discovery process in identifying potential new host defense peptides.
Project description:Routine monitoring of contaminant levels in wildlife is important for understanding chemical exposure and ultimately the link to ecosystem and human health. This is particularly important when the monitored species is recreationally hunted for human consumption. In the southeastern United States, recreational alligator harvesting takes place annually and in locations that are known to be contaminated with environmental pollutants. In this study, we investigated the biodistribution of trace elements in the American alligator (Alligator mississippiensis) from five sites in Florida, USA. These sites are locations where annual recreational alligator harvesting is permitted and two of the sites are identified as having high mercury contamination with human consumption advisories in effect. We utilized routinely collected monitoring samples (blood and scute), a commonly consumed tissue (muscle), and a classically analyzed tissue for environmental contaminants (liver) to demonstrate how the trace elements were distributed within the American alligator. We describe elemental tissue compartmentalization in an apex predator and investigate if noninvasive samples (blood and scute) can be used to estimate muscle tissue concentrations for a subset of elements measured. We found significant correlations for Hg, Rb, Se, Zn and Pb between noninvasive samples and consumed tissue and also found that Hg was the only trace metal of concern for this population of alligators. This study fills a gap in trace elemental analysis for reptilian apex predators in contaminated environments. Additionally, comprehensive elemental analysis of routinely collected samples can inform biomonitoring efforts and consumption advisories.
Project description:The muscles that effect lung ventilation are key to understanding the evolutionary constraints on animal form and function. Here, through electromyography, we demonstrate a newly discovered respiratory function for the iliocostalis muscle in the American alligator ( Alligator mississippiensis). The iliocostalis is active during expiration when breathing on land at 28°C and this activity is mediated through the uncinate processes on the vertebral ribs. There was also an increase in muscle activity during the forced expirations of alarm distress vocalizations. Interestingly, we did not find any respiratory activity in the iliocostalis when the alligators were breathing with their body submerged in water at 18°C, which resulted in a reduced breathing frequency. The iliocostalis is an accessory breathing muscle that alligators are able to recruit in to assist expiration under certain conditions.