Biology subjects

Hammerschmid, D.

Publications and source records attributed to Hammerschmid, D..

5 recordsLinked to original sources

bioRxiv2026

LemonCatcher Acidic Pull-Down Enables Selective In-Cell Hydrogen-Deuterium Exchange Mass Spectrometry

Proteins are dynamic molecules which sensitively adapt according to their environment. Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS) provides unique insights into protein conformational processes. However, existing methodology cannot selectively enrich proteins post-labeling because D-to-H back exchange must be minimized by rapid processing at pH 2.3-3.0 and 0 {degrees}C, where affinity purification fails. Here, we create LemonCatcher, a protein superglue that spontaneously forms an amide bond to the LemonTag peptide under these harsh acidic and cold quench conditions, even at -20 {degrees}C. Engineering of a bead-coupled LemonCatcher purification system introduces fast and selective quench-capture HDX-MS (SelQueX) on LemonTagged fusion proteins. We demonstrate targeted measurement of protein dynamics in living bacterial cells, revealing ligand-induced conformational changes in maltose-binding protein. Moreover, probing a stalled membrane protein nascent-chain supports a role for the ribosome in maintaining partially unfolded folding intermediates. Thus, SelQueX makes possible selective characterization of protein structural dynamics within the complex cellular milieu.

biochemistry↗

bioRxiv2026

Ionic strength modulates structural disorder and protein oligomerization in the marginally disordered Phd transcription factor

Some proteins combine sequence features that are typical both for folded proteins and intrinsically disordered proteins (IDPs). The borderline properties of these so-called "marginal" IDPs render their conformational ensembles highly sensitive to the environmental changes, which may be important for their function. Here, we investigate the prokaryotic transcription factor Phd, which regulates the phd-doc toxin-antitoxin module through an allosteric mechanism involving disorder-order transition. Using an ensemble of biophysical techniques, we show that the protein is completely disordered at low ionic strength, whereas increasing salt concentration promotes its collapse into a partially ordered monomeric state, followed by the formation of a structured dimer. Using a thermodynamic model, we decipher the linkage between ionic strength, protein stability, oligomer state and degree of disorder. Via small-angle X-ray scattering we derive the structural ensemble of dimeric Phd, revealing a gradation of disorder as a function of salt. The sequence and biophysical properties of Phd position it at the boundary between macroscopically distinct conformational ensembles, representing a large pool of states capable of engaging in functional disorder-to-order interactions, enabling Phd to act as conformational rheostat. Together with previous crystallographic data, this charts the full spectrum of disorder-to-order states in the bacterial transcription factor and underscores the structural plasticity of IDPs with the marginal sequence properties. SIGNIFICANCEWhile globular proteins adopt stable three-dimensional structures, intrinsically disordered proteins (IDPs) remain flexible and dynamically sample partially ordered states. This behavior is largely determined by amino acid composition; however, some proteins lie at the borderline between order and disorder. Here, we focus on such a protein, the Phd transcription, and show how its conformation changes from completely disordered to fully ordered. These transitions are modulated by ionic strength and binding to macromolecules, including homodimerization. Using a thermodynamic model, we map the Phd conformational space, revealing a broad ensemble of states with varying degrees of disorder. The borderline amino acids properties enable Phd to function as a conformational rheostat, coupling functional interactions to the series of graded order-disorder transitions.

biophysics↗

bioRxiv2026

Cathepsin-dependent amyloid formation drives mechanical rupture of lysosomal membranes

Lysosomal membrane integrity is essential for cellular homeostasis, and its failure drives lysosomal storage disorders (LSD) and neurodegeneration. The dipeptide L-leucyl-L-leucine methyl ester (LLOMe) is widely used to model lysosomal damage, yet its mechanism remains poorly understood. The prevailing view holds that LLOMe polymerizes into membrane-permeabilizing peptide chains within the lysosomal lumen. Using cryo-electron tomography in cultured cells and primary neurons, we visualized the structural basis of LLOMe-induced lysosomal damage. We reveal that LLOMe forms amyloid structures within lysosomes that directly interact with and rupture the limiting membrane through mechanical stress. In vitro reconstitution confirms this amyloid-mediated mechanism. These findings establish a structural paradigm for lysosomal membrane disruption and provide insights into how disease-relevant protein aggregates, implicated in neurodegeneration and LSD, may compromise lysosomal integrity.

cell biology↗

bioRxiv2024

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

biochemistry↗

bioRxiv2022

Conformational restriction shapes inhibition of a multidrug efflux adaptor protein

Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and {beta} domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

biochemistry↗