Biology subjects

Pascal, B. D.

Publications and source records attributed to Pascal, B. D..

4 recordsLinked to original sources

bioRxiv2025

HXMS: a standardized file format for HX/MS data

MotivationHydrogen/deuterium exchange-mass spectrometry (HX-MS) is a rapidly expanding technique used to investigate protein conformational ensembles. The growing popularity and utility of HX-MS has driven the development of diverse instrumentation and software, resulting in inconsistent, non-standardized data analysis and representation. Most HX-MS data formats also employ only mean deuteration representations of the data rather than full isotopic mass spectra, which reduces the information content of the data and limits downstream quantitative analysis. ResultsInspired by reliable protein structure and genomics data formats, we present HXMS, a unified, lightweight, scalable, and human-readable file format for HX-MS data. The HXMS format preserves the isotopic mass envelopes for all peptides, captures the full experimental time-course including fully deuterated control samples, and contains all other key information. It supports multimodal distributions, post-translational modifications (PTMs), and experimental replicates. To promote compatibility with existing HX-MS workflows, we also developed PFLink, a Python package that converts exported data files from commonly used HX-MS software to the HXMS format. PFLink and the HXMS format will enable more quantitative, higher-resolution data processing, improved data sharing and storage among HX-MS practitioners, future machine learning applications, and further developments in HX-MS analysis. Availability and implementationPFLink is publicly available to install locally on HuggingFace, alongside documentation, or use online at HuggingFace (https://huggingface.co/spaces/glasgow-lab/PFlink). The supplemental information includes sample input files, sample HXMS files, and a generic unfilled PFlink custom CSV file that users may populate with key experimental conditions and results, which can then be read and converted into the HXMS format.

biophysics↗

bioRxiv2025

A ternary switch determines ERα LBD conformation

The transcription factor estrogen receptor (ER) is the primary driver of ER+ breast cancer progression and a target of multiple FDA-approved anticancer drugs. Ligand-dependent activity of ER is determined by the conformation of helix-12 (H12) within the ligand binding domain (LBD), but how H12 transitions from an unliganded (apo) state to active (estrogen-bound) or inactive (SERM/SERD-bound) states remains unresolved. Here, we present the first crystal structure of an apo ER LBD, revealing a third distinct H12 conformation that regulates receptor activity. Structural mass-spectrometry, small-angle X-ray scattering, functional analysis and molecular dynamics simulations reveal that the apo conformation of H12 is stable in the absence of ligand, but is destabilised by Y537S and D538G breast cancer mutations driving constitutive activation. We propose a model in which H12 functions as a ternary molecular switch to determine receptor activity. These findings provide critical insights into the ligand-dependent and -independent regulation of ER and have significant implications for therapeutic intervention.

biochemistry↗

bioRxiv2024

Structural proteomics defines a sequential priming mechanism for the progesterone receptor

The progesterone receptor (PR) is a steroid-responsive nuclear receptor with two isoforms: PR-A and PR-B. Disruption of PR-A:PR-B signaling is associated with breast cancer through interactions with oncogenic co-regulatory proteins (CoRs). However, molecular details of isoform-specific PR-CoR interactions remain poorly understood. Using structural mass spectrometry, we investigate the sequential binding mechanism of purified full-length PR and intact CoRs, steroid receptor coactivator 3 (SRC3) and p300, as complexes on target DNA. Our findings reveal selective CoR NR-box binding by PR and unique interaction surfaces between PR and CoRs during complex assembly, providing a structural basis for CoR sequential binding on PR. Antagonist-bound PR showed persistent CoR interactions, challenging the classical model of nuclear receptor activation and repression. Collectively, we offer a peptide-level perspective on the organization of the PR transcriptional complex and infer the mechanisms behind the interactions of these proteins, both in active and inactive conformations.

biochemistry↗

bioRxiv2018

Disorganization of the histone core promotes organization of heterochromatin into phase-separated droplets

The heterochromatin protein HP1 is proposed to enable chromatin compaction via liquid droplet formation. Yet, a connection between phase separation and chromatin compaction has not been experimentally demonstrated. More fundamentally, how HP1 action at the level of a single nucleosome drives chromatin compaction remains poorly understood. Here we directly demonstrate that the S. pombe HP1 protein, Swi6, compacts arrays of multiple nucleosomes into phase-separated droplets. Using hydrogen-deuterium exchange, NMR, and mass-spectrometry, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a mononucleosome. Restraining these dynamics via site-specific disulfide bonds impairs the compaction of nucleosome arrays into phase-separated droplets. Our results indicate that chromatin compaction and phase separation can be highly coupled processes. Further, we find that such coupling is promoted by a counter-intuitive function of Swi6, namely disorganization of the octamer core. Phase separation is canonically mediated by weak and dynamic multivalent interactions. We propose that dynamic exposure of buried histone residues increases opportunities for multivalent interactions between nucleosomes, thereby coupling chromatin compaction to phase separation. We anticipate that this new model for chromatin organization may more generally explain the formation of highly compacted chromatin assemblies beyond heterochromatin.

biophysics↗