Biology subjects

Wells, M. L.

Publications and source records attributed to Wells, M. L..

6 recordsLinked to original sources

bioRxiv2026

Simultaneous single-cell profiling of the transcriptome and proteome

Transcriptomic and proteomic measurements from the same single cell provide complementary information that cannot be inferred from either modality alone, yet methods for the parallel recovery of both analyte classes from a single-cell lysate remain limited. Here, we describe a workflow in which individual cells are isolated by automated dispensing into a minimal, MS-compatible lysis volume, followed by sequential mRNA capture and protein supernatant recovery, prior to independent downstream processing. The method is compatible with standard library preparation and data-independent acquisition proteomics pipelines and requires no dedicated instrumentation beyond a single-cell dispensing platform. We evaluated workflow performance on 67 single cells across 3 iBlastoids. Transcriptomic sequencing detected a median of 5375 genes per cell, and proteomic analysis identified a median of 2123 protein groups per cell across two mass spectrometry platforms. Compared with a standalone single-cell proteomics protocol, incorporating the mRNA extraction step reduced median proteomic depth by approximately 11% (median 1,965 vs. 2,204 protein groups per cell), while mean per-cell identification remained comparable across workflows (1,790 vs. 1,775 protein groups per cell). Direct comparison of paired transcript and protein abundance yielded a median Spearman correlation of {rho} {approx} 0.38; after correction for detection depth, the partial correlation was 0.067.

systems biology↗

bioRxiv2026

Bidirectional allosteric ligand regulation in a central glycolytic enzyme

Allosteric regulation enables fine-tuned control of enzyme activity in response to cellular signals, yet its molecular basis often remains unclear. Phosphofructokinase-1 (PFK), the rate-limiting enzyme of glycolysis, is a paradigmatic, well-conserved system whose reaction kinetics conform to the Monod-Wyman-Changeux model of allostery. However, X-ray crystal structures of bacterial PFK orthologs in distinct ligand-bound states do not show the consistent, concerted structural rearrangements expected for classical "relaxed" and "tense" states, revealing a decades-long disconnect between structure and function. We resolve this paradox by integrating biophysical and computational approaches to show that activator and inhibitor binding to the same allosteric pocket differentially reweight the conformational ensemble of Escherichia coli PFK. Activator binding stabilizes conformational substates that preorganize the catalytic site, whereas inhibitor binding upweights apo-like, catalytically incompetent substates. These findings establish an ensemble-based mechanism for PFK regulation and provide an energetic framework for understanding the expanded allosteric architecture of higher PFK orthologs.

biophysics↗

bioRxiv2025

The duration of nutrient limiting conditions can contribute to shaping subsequent diatom community composition; insights from laboratory experiments

Climate-driven increases in global surface water temperatures are enhancing upper ocean stratification likely resulting in more prolonged periods of nutrient limitation. Although nutrient limitation in diatoms and their growth responses to increasing temperatures have been studied extensively, much less is known about their growth response to nutrient injection after prolonged durations of nutrient limitation. This study examines the growth response of three bloom-forming diatom species: Pseudo-nitzschia pungens, P. australis, and Skeletonema costatum after short-term ([~]2 week) and prolonged ([~]4 week) periods of nutrient limitation at five temperatures (9, 12, 15, 20, and 25{degrees}C). Pseudo-nitzschia species showed shorter lag times and higher growth rates than S. costatum after prolonged nutrient stress. These findings demonstrate that certain diatom species can exhibit faster growth recovery after prolonged nutrient limitation and in warmer conditions compared to others, providing new insights on drivers that shape phytoplankton communities.

ecology↗

bioRxiv2025

A Hotspot Phosphorylation Site on SHP2 Drives Oncoprotein Activation and Drug Resistance

SHP2 is a phosphatase and a critical mediator of receptor tyrosine kinase (RTK)-driven RAS/mitogen-activated protein kinase (MAPK) signaling. Despite promising preclinical data, SHP2 inhibitors have shown minimal clinical efficacy, with no defined clinical mechanisms of primary resistance. Here, we elucidate phosphorylation of SHP2 at tyrosine 62 (pY62) as a hotspot phosphorylation site in the proteome and RTK-driven tumor types in patients. We demonstrate that SRC family kinases directly phosphorylate SHP2 at Y62, downstream of but not directly phosphorylated by RTKs. Using biochemical and biophysical analyses, we show that SHP2 Y62D enforces an open, active conformation, resulting in constitutive phosphatase activation that is sufficient to activate MAPK signaling and confer resistance to allosteric SHP2 inhibitors. These findings establish that SHP2 pY62 is a phosphorylation hotspot phenocopying mutational activation, a mechanism of primary resistance to SHP2 inhibitors, and a cancer drug target distinct from wildtype SHP2. Statement of significanceThis study identifies phosphorylation of SHP2 at tyrosine 62 (pY62) as a conserved mechanism of resistance to allosteric SHP2 inhibitors. By stabilizing an open, active SHP2 conformation, pY62 phenocopies oncogenic PTPN11 mutations and sustains MAPK signaling across cancer types. These findings redefine SHP2 inhibitor resistance as a phosphorylation-driven, target-intrinsic process, nominate pY62 as a potential biomarker for therapeutic response, and propose phosphorylated SHP2 as a distinct drug target.

cancer biology↗

bioRxiv2025

Conserved energetic changes drive function in an ancient protein fold

While the ongoing revolution in structural biology offers an unprecedented understanding of the relationship between protein structure and function, it also confirms a puzzling, widely applicable principle: protein domains with highly conserved three-dimensional folds can perform radically disparate biochemical functions. To gain insight to this fundamental structural enigma, we mapped the energetic landscapes of a family of bacterial transcription factors and their anciently diverged structural homologs, the periplasmic binding proteins. Using hydrogen exchange/mass spectrometry, bioinformatics, X-ray crystallography, and molecular dynamics, we uncovered an unexpected contrast: despite binding the same sugars, the two families evolved unique "energetic blueprints" to support their distinct functional requirements. To test if differences in energetic ensembles have functional consequences, we rationally redesigned the protein fold for tunable ligand-driven transcriptional responses. Strikingly, energy-driven protein engineering produced synthetic transcription factors with the theoretically anticipated ligand-induced transcriptional outputs. Thus, decoding energetic blueprints among conserved protein folds provides a novel explanation for diverse functional adaptations, paves an alternative roadmap for protein design, and offers a new approach for engineering challenging drug targets.

biophysics↗

bioRxiv2024

Site-resolved energetic information from HX/MS experiments

High-resolution energetic information about protein conformational ensembles is essential for understanding protein function, yet remains challenging to obtain. We present PIGEON-FEATHER, a method for calculating ensemble free energies of opening ({Delta}Gop) at single- or near-single-amino acid resolution for proteins of all sizes from hydrogen exchange/mass spectrometry (HX/MS) data. PIGEON-FEATHER disambiguates and reconstructs all experimentally measured HX/MS isotopic mass envelopes using a Bayesian Monte Carlo sampling approach. We applied PIGEON-FEATHER to reveal how E. coli and human dihydrofolate reductases (ecDHFR, hDHFR) have evolved distinct ensembles. We show how two competitive inhibitors bind these orthologs differently, solving the longstanding mystery of why both therapeutic molecules inhibit hDHFR, but only one inhibits ecDHFR. Extending PIGEON-FEATHER to a large protein-DNA complex, we mapped ligand-induced ensemble reweighting in the E. coli lac repressor to describe the functional switching mechanism crucial for transcriptional regulation.

biophysics↗