Biology subjects

Horner, A.

Publications and source records attributed to Horner, A..

5 recordsLinked to original sources

bioRxiv2025

A conserved cytoplasmic glutamic acid mediates pH-dependent gating in bacterial urea channel UreI

The bacterial UreI channel family enables rapid urea uptake, essential for urease activity and acid resistance. While pH-dependent gating in Helicobacter pylori UreI (HpUreI) is attributed to periplasmic histidines, the role of cytoplasmic residues remains unexplored. The UreI homolog from Streptococcus salivarius (SsUreI), lacking periplasmic histidines, serves as a simplified model to identify gating determinants. Here, we combine yeast complementation assays, in vitro studies, and MD simulations to show that cytoplasmic glutamic acid E136 mediates pH-dependent gating in SsUreI. Mutagenesis in H. pylori/H. hepaticus homologs confirm E136 as a conserved pH sensor across UreI channels. Protonation of E136 disrupts its salt bridge with R20, increasing cytoplasmic-loop flexibility and urea-permeable filter conformations. These findings challenge the paradigm of exclusive periplasmic pH sensing, supporting a Gram-negative-specific dual-sensor model (E136 + histidines). By elucidating this molecular mechanism, we identify E136 as a therapeutic target to disrupt UreI-mediated acid resistance in pathogenic bacteria.

biophysics↗

bioRxiv2025

Clear Native Gel Electrophoresis for the Purification of Fluorescently Labeled Membrane Proteins in Native Nanodiscs

Native gel electrophoresis techniques, such as blue or clear native gel electrophoresis (BNE or CNE), are widely used to separate and characterize proteins. However, in high-resolution CNE, mild anionic or neutral detergents are often used at concentrations too low to prevent membrane-protein aggregation. Additionally, the identification of proteins is hampered by the lack of suitable molecular-weight markers, like those used in SDS-PAGE. Here, we introduce a novel approach that combines charged polymer-encapsulated nanodiscs and fluorescence correlation spectroscopy (FCS) to address both challenges. Membrane proteins are first extracted using Glyco-DIBMA, a negatively charged amphiphilic copolymer. This enables the spontaneous formation of nanodiscs harboring the fluorescently labeled target protein within a native-like lipid-bilayer environment, which is confirmed by FCS. The nanodiscs are then subjected to detergent-free CNE. As the number of protomers increases, the nanodiscs grow larger resulting in increased migration distances in CNE due to higher charge densities. Crucially, the nanodiscs remain intact throughout CNE, as demonstrated by FCS analysis of resolubilized bands excised from the gels. Moreover, the membrane proteins used in this study: a potassium channel (KvAP), a sodium channel (NavMs), a water channel (GlpF), and a urea channel (HpUreI) do not aggregate, as shown by the fluorescent brightnesses and diffusion times of individual nanodiscs. Moreover, the oligomeric states of membrane proteins can be deduced from the brightness per nanodisc. Since purified membrane proteins remain within a native-like lipid-bilayer environment and avoid detergent exposure, they are immediately suitable for downstream structural and functional studies.

biophysics↗

bioRxiv2022

The hidden intricacies of aquaporins: Remarkable details in a common structural scaffold

Evolution turned aquaporins (AQPs) into the most efficient facilitators of passive water flow through cell membranes at no expense of solute discrimination. In spite of a plethora of solved AQP structures, many structural details remain hidden. Here, by combining extensive sequence- and structural-based analysis of a unique set of 20 non-redundant high-resolution structures and molecular dynamics simulations of 4 representatives, we identify key aspects of AQP stability, gating, selectivity, pore geometry and oligomerization, with a potential impact on channel functionality. We challenge the general view of AQPs possessing a continuous open water pore and depict that AQPs selectivity is not exclusively shaped by pore lining residues but also by the relative arrangement of transmembrane helices. Moreover, our analysis reveals that hydrophobic interactions constitute the main determinant of protein thermal stability. Finally, we establish a novel numbering scheme of the conserved AQP scaffold facilitating direct comparison and prediction of potential structural effects of e.g. disease-causing mutations. Additionally, our results pave the way for the design of optimized AQP water channels to be utilized in biotechnological applications.

bioinformatics↗

bioRxiv2019

Binding of the motor protein SecA to the bacterial protein translocation channel SecYEG in the absence of ATP

Translocation of many secretory proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG-SecA complex that differ in the protrusion depth of SecAs two-helix finger into SecYEGs aqueous channel. Such movement of the finger is in line with the power stroke mechanism of protein translocation.

biophysics↗

bioRxiv2017

No effect of hippocampal lesions on stimulus-response bindings

The hippocampus is believed to be important for rapid learning of arbitrary stimulus-response contingencies, or S-R bindings. In support of this, Schnyer et al., 2006 (Experiment 2) measured priming of reaction times (RTs) to categorise visual objects, and found that patients with medial temporal lobe damage, unlike healthy controls, failed to show evidence of reduced priming when response contingencies were reversed between initial and repeated categorisation of objects (a signature of S-R bindings). We ran a similar though extended object classification task on 6 patients who appear to have selective hippocampal lesions, together with 24 age-matched controls. Unlike Schnyer et al., 2006, we found that reversing response contingencies abolished priming in both controls and patients. Bayes Factors provided no reason to believe that response reversal had less effect on patients than controls. We therefore conclude that it is unlikely that the hippocampus is needed for S-R bindings.

neuroscience↗