Biology subjects

Barran, P. E.

Publications and source records attributed to Barran, P. E..

6 recordsLinked to original sources

bioRxiv2026

Adaptation to free-living drives loss of beneficial endosymbiosis through metabolic trade-offs

Symbioses are widespread (1) and underpin the function of diverse ecosystems (2-6), but their evolutionary stability is challenging to explain (7,8). Fitness trade-offs between contrasting intracellular and extracellular niches could act to stabilise endosymbioses because adaptation to either niche is predicted to reduce fitness in the alternate niche, thus reinforcing symbiosis (8,9). Here, we experimentally evolved four diverse Chlorella green algal endosymbionts of Paramecium bursaria to free-living conditions supplying either an amino acid, as provisioned by hosts (10,11), or nitrate, as available in freshwater (12), as the sole nitrogen source. Experimental algal populations adapted to free-living environments, generally increasing in population density and cellular chlorophyll content over time. In one of the four endosymbiont strains, adaptation to the nitrate free-living environment, but not the amino acid environments, drove the loss of fitness benefits to the host in reconstituted symbioses. This loss was not associated with reduced ability to grow on host-provisioned amino acids, nor lost ability to release the sugars provisioned to the host (10,13). Genome sequencing of evolved algal lines revealed genomic divergence between nitrate-adapted and amino acid-adapted lines, affecting genes involved with metabolic organisation and intracellular resource transport. Untargeted metabolomic profiling further showed extensive changes to membrane remodelling and turnover in N-evolved lines. Together, our data support a role for metabolic trade-offs driving divergence between contrasting intracellular and extracellular niches, with nitrogen as a key environmental axis driving divergence. Fitness trade-offs may, therefore, be a general, simple mechanism acting to reinforce symbiosis, contributing to evolutionary stability.

evolutionary biology↗

bioRxiv2025

Triplet Quenching by Active Site Cysteine Residues Improves Photostability in Fatty Acid Photodecarboxylase

Enzyme photobiocatalysis uses light to drive high-energy transformations but is limited by the rarity of photoenzymes. Fatty acid photodecarboxylase (FAP), a recently discovered photoenzyme, enables fatty acid conversion to alkanes/alkenes via excitation of an FAD cofactor, though its poor photostability and photoinactivation has hindered industrial applications. Here, we combine protein engineering approaches with biocatalytic and biophysical techniques, as well as computational chemistry, to demonstrate that additional active site cysteine residues can suppress oxygen-mediated inactivation processes that are driven by the FAD triplet-excited state. We identify a number of positions close to the FAD for cysteine residues that lead to a significant enhancement in activity as a result of an increase in the number of catalytic turnovers and improved photostability. The additional cysteine residues quench the triplet excited state of the FAD cofactor via a proposed proton-coupled electron transfer mechanism, resulting in lower levels of harmful reactive oxygen species. Our study highlights promising routes to mitigate non-productive, photoinactivation pathways in FAP and informs the rational design of new flavin-based photoenzymes.

biochemistry↗

bioRxiv2024

Efficient overexpression and purification of SARS-CoV-2 Nucleocapsid proteins in Escherichia coli

The fundamental biology of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.

biochemistry↗

bioRxiv2018

Novel allosteric mechanism of p53 activation by small molecules for targeted anticancer therapy

Given the immense significance of p53 restoration for anti-cancer therapy, elucidation of the mechanisms of action of p53-activating molecules is of the utmost importance. Here we report a discovery of novel allosteric modulation of p53 by small molecules, which is an unexpected turn in the p53 story. We identified a structural element involved in p53 regulation, whose targeting by RITA, PpIX and licofelone block the binding of p53 inhibitors, MDM2 and MDMX. Deletion and mutation analysis followed by molecular modeling, identified the key p53 residues S33 and S37 targeted by RITA and PpIX. We propose that the binding of small molecules to the identified site induces a conformational trap preventing p53 from the interaction with MDM2 and MDMX. These results point to a high potential of allosteric activators. Our study provides the basis for the development of therapeutics with a novel mechanism of action, thus extending the p53 pharmacological potential.

cancer biology↗

bioRxiv2018

Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation

Allosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cellular metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, it remains elusive how allosteric inputs upon simultaneous binding of different ligands are integrated to regulate PKM2 activity. Here, we show that, in the presence of the allosteric inhibitor L-phenylalanine (Phe), the activator fructose 1,6-bisphosphate (FBP) can induce PKM2 tetramerisation, but fails to maximally increase enzymatic activity. Guided by a new computational framework we developed to identify residues that mediate FBP-induced allostery, we generated two PKM2 mutants, A327S and C358A, in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings demonstrate a role for residues involved in FBP-induced allostery in enabling the integration of allosteric input from Phe and reveal a mechanism that underlies the co-ordinate regulation of PKM2 activity by multiple allosteric ligands.

biochemistry↗

bioRxiv2017

A Careful Consideration of the Influence of Structure, Partial charges and Basis Sets on Collision Cross Sections of Monosaccharides when Comparing Values from DFT Calculated Conformers to those Obtained Experimentally

Molecular modelling is routinely employed to assign 3D structures to collision cross sections (CCSs) derived from ion mobility mass spectrometry experiments (IM-MS). The assignment of model structures to the experimental CCSs remains an ambiguous task, where one of several methods may be used to obtain a CCS from a given set of coordinates. The most reliable of the commonly used techniques, the Trajectory Method, starts with atomic coordinates which can be accompanied by partial atomic charges, obtained using ab initio methods. Here, we use lithiated - and {beta}-glucose ions as exemplar molecules to detect the effect conformational modification and changes to the partial charge distribution have on computed collision cross sections. Six popular charge schemes (Mulliken, APT, CHelpG, MK, HLY and NPA) were examined in combination with three functionals (Hartree-Fock, B3LYP and M05) and five basis sets (STO-3G, 3-21G, 6-31G, 6-31+G and 6-31G*) on twenty unique structures. Our findings indicate that molecular conformation makes a significant contribution to fluctuations of partial charges in Electrostatic Potential (ESP) and Mulliken charge scheme; Partial charges derived using Natural Population Analysis (NPA) and ESP methods are largely independent of functional and basis set selection; and both selection of the charge scheme and functional/basis set combination play a large role in the resultant CCS, often causing few percent fluctuations in the computed values.

biochemistry↗