Biology subjects

Wells, S. A.

Publications and source records attributed to Wells, S. A..

5 recordsLinked to original sources

bioRxiv2026

Fecal Hormone Metabolites Concentrations Collected Longitudinally Confirm Spontaneous Polyestry in Mt. Graham Red Squirrels (Tamiasciurus fremonti grahamensis)

The Mount Graham red squirrel (Tamiasciurus fremonti grahamensis, MGRS) is a federally endangered subspecies endemic to southeastern Arizona. Despite the establishment of ex situ conservation programs, reproduction outside the wild has not occurred. We monitored fecal hormone metabolites (FHM) to noninvasively assess estrous cycling and reproductive hormone dynamics in ex situ-managed MGRS. We compared ex situ findings with two in situ (wild) populations. We tracked longitudinal changes in estradiol and progesterone metabolites in three ex situ managed females and evaluated whether ovulation was spontaneous or induced. We detected 22 ovulation events over two years, with timing and regularity consistent with spontaneous polyestrous cycling. Comparative hormone analyses revealed appreciably lower progesterone and corticosteroid metabolite levels in ex situ individuals, whereas estradiol levels were comparable to those of wild counterparts. These findings suggest that maintaining environmental, and social conditions, e.g., lighting, temperature, and conspecific proximity, in ex situ settings is critical and could influence reproductive success. By confirming spontaneous ovulatory cycling in MGRS and demonstrating the utility of FHM as a noninvasive reproductive assessment tool, this study advances ex situ breeding efforts and provides a framework for reproductive monitoring in other endangered taxa.

physiology↗

bioRxiv2022

N-linked glycosylation increases horse radish peroxidase rigidity leading to enhanced activity and stability.

Glycosylation is the most prevalent protein post-translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet the full impact of glycosylation on the protein structure-function relationship, especially in enzymes, is still limited. Here we show glycosylation rigidifies the important commercial enzyme horseradish peroxidase (HRP), which in turn increases its activity and stability. Circular dichroism spectroscopy revealed that glycosylation increased holo-HRPs thermal stability and promoted significant helical structure in the absence of haem (apo-HRP). Glycosylation also resulted in a 10-fold increase in enzymatic turnover towards o-phenylenediamine dihydrochloride when compared to its non-glycosylated form. Utilising a naturally occurring site-specific probe of active site flexibility (Trp117) in combination with red-edge excitation shift fluorescence spectroscopy, we found that glycosylation significantly rigidified the enzyme. In silico simulations confirmed that glycosylation largely decreased protein backbone flexibility, especially in regions close to the active site and the substrate access channel. Thus, our data shows that glycosylation does not just have a passive effect on HRP stability but can exert long range effects that mediate the native enzymes activity and stability through changes in inherent dynamics.

biochemistry↗

bioRxiv2021

Structure and dynamics of a cold-active esterase reveals water entropy and active site accessibility as the likely drivers for cold-adaptation

Cold-active esterases hold great potential for undertaking useful biotransformations at low temperatures. Here, we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical /{beta} hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7s closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Comparison of B-factors with the closest related mesophilic and thermophilic esterases suggests the EstN7 cap region is proportionally less flexible. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are the main drivers of EstN7s cold adaptation rather than changes in dynamics.

biochemistry↗

bioRxiv2020

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a fingerprint of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (g-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

biochemistry↗

bioRxiv2020

Rigidity, normal modes and flexible motion of a SARS-CoV-2 (COVID-19) protease structure.

The rigidity and flexibility of two recently reported crystal structures (PDB entries 6Y2E and 6LU7) of a protease from the SARS-CoV-2 virus, the infectious agent of the COVID-19 respiratory disease, has been investigated using pebble-game rigidity analysis, elastic network model normal mode analysis, and all-atom geometric simulations. This computational investigation of the viral protease follows protocols that have been effective in studying other homodimeric enzymes. The protease is predicted to display flexible motions in vivo which directly affect the geometry of a known inhibitor binding site and which open new potential binding sites elsewhere in the structure. A database of generated PDB files representing natural flexible variations on the crystal structures has been produced and made available for download from an institutional data archive. This information may inform structure-based drug design and fragment screening efforts aimed at identifying specific antiviral therapies for the treatment of COVID-19.

molecular biology↗