Biology subjects

Jones, B. J.

Publications and source records attributed to Jones, B. J..

6 recordsLinked to original sources

bioRxiv2025

Measurement and Control of Crossed Potentials in a Flavoprotein

Flavoproteins are versatile redox-active biomolecules enabling a multitude of metabolic processes. Their versatility stems from the tunability of the flavin cofactors one- and two-electron reduction potentials via interactions with the protein scaffold, which have dramatic influence on reactivity. Although several mechanisms have been proposed to explain how the flavin-binding pocket modulates redox thermodynamics, few have been validated through quantitative experiments. In this study, we investigate how the flavin N5 environment influences the redox properties of the flavin mononucleotide cofactor in the "improved" light-oxygen-voltage (iLOV) sensing protein using site-directed mutagenesis, redox titrations, and hybrid quantum mechanical molecular mechanical (QM/MM) methods combined with classical alchemical free energy simulations. Mutating the residue Q103, which interacts with the flavin N5 and O4' atoms in the X-ray crystallographic structure, exerts a modest < 35 mV effect on the overall two-electron reduction potential, but significantly alters the potential separation of the two one-electron couples (potential crossing) by up to 168 mV. QM/MM and free energy calculations reveal that water penetration into the flavin binding pocket near N5 and O4' largely explains the trend in reduction potentials among the mutants. The results suggest a molecular mechanism of flavin tuning in which hydrogen bonding to the neutral semiquinone, either directly by the side-chain or a protein-penetrating water, contributes significantly to the potential crossing. These findings establish quantitative experimental benchmarks for theoretical models and advance a molecular mechanism for redox tuning in flavoproteins.

biochemistry↗

bioRxiv2023

Insulin-mediated endothelin signaling is antiviral during West Nile virus infection

West Nile virus (WNV) is the most prevalent mosquito-borne virus in the United States with approximately 2,000 cases each year. There are currently no approved human vaccines and a lack of prophylactic and therapeutic treatments. Understanding host responses to infection may reveal potential intervention targets to reduce virus replication and disease progression. The use of Drosophila melanogaster as a model organism to understand innate immunity and host antiviral responses is well established. Previous studies revealed that insulin-mediated signaling regulates WNV infection in invertebrates by regulating canonical antiviral pathways. Because insulin signaling is well-conserved across insect and mammalian species, we sought to determine if results using D. melanogaster can be extrapolated for the analysis of orthologous pathways in humans. Here, we identify insulin-mediated endothelin signaling using the D. melanogaster model and evaluate an orthologous pathway in human cells during WNV infection. We demonstrate that endothelin signaling reduces WNV replication through the activation of canonical antiviral signaling. Taken together, our findings show that endothelin-mediated antiviral immunity is broadly conserved across species and reduces replication of viruses that can cause severe human disease. IMPORTANCEArboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. Here, we identify a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling. Additionally, we demonstrate that we can successfully translate results obtained from D. melanogaster into the more relevant human system. Our results add to the growing field of insulin-mediated antiviral immunity and identifies potential biomarkers or intervention targets to better address West Nile virus infection and severe disease.

microbiology↗

bioRxiv2022

Wheat pollen uptake of CRISPR/Cas9 RNP-PDMAEMA nanoassemblies results in targeted loss of gene function in progeny

The utility of CRISPR in plants has remained limited by the dual difficulties of delivering the molecular machinery to target cells and the use of somatic cell techniques that require tissue culture-based de novo organogenesis. We developed 5-10 nm isodiametric polyplex nanoassemblies, comprising poly [2-(dimethylamino)ethylmethacrylate] PDMAEMA (PD) polycationic linear homopolymers and CRISPR/Cas9 ribonucleoproteins (RNPs), that enable endocytosis-driven RNP uptake into pollen grains. Pollen from wheat plants (genotype Gladius+Sr50), homozygous for monogenic Sr50-mediated resistance to stem rust (Puccinia graminis f. sp. tritici -Pgt), were incubated with RNP/PD nanoassemblies targeting the dominant, Sr50 rust resistance gene. The treated pollen grains were then used to fertilize Gladius+Sr50 florets and the resulting M1 plants were tested for loss of Sr50 function via rust resistance screens. The identification of fully susceptible M1 seedlings indicated that the Sr50 RNPs acted on both alleles, indicating they were transferred via the treated pollen to the zygote. The ability to readily deliver CRISPR RNPs to reproductive cells via biodegradable, polymeric nanocomplexes has significant implications for the efficiency of gene editing in plants.

plant biology↗

bioRxiv2020

Consensus Finder web tool to predict stabilizing substitutions in proteins

The consensus sequence approach to predicting stabilizing substitutions in proteins rests on the notion that conserved amino acids are more likely to contribute to the stability of a protein fold than non-conserved amino acids. To implement a prediction for a target protein sequence, one finds homologous sequences and aligns them in a multiple sequence alignment. The sequence of the most frequently occurring amino acid at each position is the consensus sequence. Replacement of a rarely occurring amino acid in the target with a frequently occurring amino acid is predicted to be stabilizing. Consensus Finder is an open-source web tool that automates this prediction. This chapter reviews the rationale for the consensus sequence approach and explains the options for fine-tuning this approach using Staphylococcus nuclease A as an example.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗

bioRxiv2019

LUXendins reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and STED-compatible LUXendin651 we describe islet GLP1R expression patterns, reveal higher-order GLP1R organization including the existence of membrane nanodomains, and track single receptor subpopulations. We furthermore show that different fluorophores can confer agonistic behavior on the LUXendin backbone, with implications for the design of stabilized incretin-mimetics. Thus, our labeling probes possess divergent activation modes, allow visualization of endogenous GLP1R, and provide new insight into class B GPCR distribution and dynamics.

cell biology↗

bioRxiv2018

Dextranol: A better lyoprotectant

Dextranol, a reduced dextran, prevents damage to stored dry protein samples that unmodified dextran would otherwise cause. Lyoprotectants like the polysaccharide dextran are critical for preserving dried protein samples by forming rigid a glass that protects entrapped protein molecules. Stably dried proteins are important for maintaining critical information in clinical samples like blood serum. However, we found that dextran reacts with serum proteins during storage, producing high-molecular weight Amadori-product conjugates. These conjugates appeared in a matter of days or weeks when stored at elevated temperatures (37{degrees} or 45{degrees}C), but also appeared on a timescale of months when stored at room temperature. We synthesized a less reactive dextranol by reducing dextrans anomeric carbon from an aldehyde to an alcohol. Serum samples dried in a dextranol- based matrix protected the serum proteins from forming high-molecular weight conjugates. The levels of four cancer-related serum biomarkers (prostate specific antigen, neuropilin-1, osteopontin, and metalloproteinase 7) decreased, as measured by immunoassay, when serum samples were stored for one to two weeks in dextran- based matrix. Switching to a dextran-based lyoprotection matrix slightly reduced the damage to osteopontin and completely stopped any detectable damage during storage in the other three biomarkers when for a period of two weeks at 45{degrees}C. Dextranol offers a small and easy modification to dextran that significantly improves the molecules function as a lyoprotectant by eliminating the potential for damaging protein-polysacharide conjugation.

biochemistry↗