Biology subjects

Sampson, C. D. D.

Publications and source records attributed to Sampson, C. D. D..

3 recordsLinked to original sources

bioRxiv2026

Structural modelling and functional analysis support lipid binding by the dimeric E. coli DedA protein YqjA

Maintenance of membrane homeostasis is essential for bacterial viability, yet the molecular functions of many membrane proteins involved in this process remain poorly understood. The widely distributed DedA superfamily of integral membrane proteins has been implicated in membrane homeostasis, with deletion of DedA genes resulting in sensitivities to temperature, pH and a range of antimicrobial compounds. Several bacterial DedA proteins have been linked to lipid transport, providing a potential connection between function and phenotype. However, a lack of direct functional and structural data means that the precise role of DedA proteins in bacterial membrane homeostasis remains unclear. Here, using analytical ultracentrifugation (AUC), we show that the Escherichia coli DedA protein YqjA exists predominantly as a dimer and, by combining structural modelling with site-specific cysteine crosslinking, we have identified the most likely dimer interface. Modelling of dimeric YqjA in the presence of lipids predicts an interfacial lipid-binding site located in close proximity to several conserved, functionally important residues. Consistent with lipid binding, addition of lipid substantially increased the thermal stability of YqjA. Furthermore, mutation of residues associated with the predicted binding site impaired YqjA function. Together, these findings provide new structural and functional insights into the DedA family and support a role for lipid binding in the activity of YqjA.

biochemistry↗

bioRxiv2020

Substrate-dependent accessibility changes in the Na+-dependent C4-dicarboxylate transporter, VcINDY, suggest differential substrate effects in a multistep mechanism.

Members of the divalent anion sodium symporter (DASS) family (SLC13 in humans) play critical roles in metabolic homeostasis, influencing many processes including fatty acid synthesis, insulin resistance, adiposity, and lifespan determination. DASS transporters catalyse the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and VcINDY conformation using a site-specific alkylation strategy to probe the solvent accessibility of several broadly distributed positions in VcINDY in the presence and absence of substrates (Na+ and succinate). Our findings reveal that accessibility to all positions tested can be modulated by the presence of substrates, with the majority becoming less accessible in the presence of Na+. Mapping these solvent accessibility changes onto the known structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified intermediate state. We also observe substantial, separable effects of Na+ and succinate binding at several amino acid positions suggesting distinct effects of the two substrates. Furthermore, analysis of a solely succinate-sensitive residue indicates that VcINDY binds its substrate with a low affinity and proceeds via an ordered process in which one or more Na+ ions must bind prior to succinate. These findings provide insight into the mechanism of VcINDY, which is currently the only structural-characterised representative of the entire DASS family.

biochemistry↗

bioRxiv2017

Codon-Dependent Translational Accuracy Controls Protein Quality in Escherichia coli but not in Saccharomyces cerevisiae

In order to generate a functional proteome, gene expression pathways must assemble proteins accurately according to the rules of the genetic code. General gene expression accuracy is known to be high, but errors nevertheless occur with measurable frequencies. Here we develop a mass-spectrometry (MS) based assay for the detection of a particular type of gene expression error, amino acid misincorporation. This assay allows assessing a much broader range of misincorporation events compared to current, very sensitive but also very specific enzyme reporter assays. Our assay uncovers a remarkably rich pool of error products for a model protein expressed in E. coli, which depend quantitatively on codon usage in the expression construct. This codon usage dependence can be explained in part as a function of the composition of the tRNA pool in this organism. We further show that codon-dependent differences in error levels correlate with measurable changes in specific protein activity. In contrast to E. coli, error levels are lower, and appear not to be codon usage dependent, when the same model protein is expressed in S. cerevisiae.

molecular biology↗