Biomedical subjects

Joseph A Loo

Publications and source records attributed to Joseph A Loo.

5 recordsLinked to original sources

PubMed2026

Solvent Leveling Explains Supercharging in Electrospray Ionization Mass Spectrometry.

Supplementing standard electrospray ionization (ESI) solvents with specific low-volatility organic compounds (e.g., sulfolane or any positional isomer of nitrobenzyl alcohol) increases biomolecular analyte charge for mass spectrometry in the phenomenon known as supercharging. Controversial mechanisms responsible for increasing charge are considered, and the data is found to correlate highly to solvent leveling; i.e., protonated solvent is the strongest acid in a solution because any stronger acid simply dissociates to protonate more solvent. Hence, the recipe for increasing charge in positive ion mode is to make the protonated solvent into a stronger acid (equivalent to reducing the neutral solvent's basicity). That change is accomplished by adding involatile, weak bases to the solvent. A secondary effect of weak base additives is to suppress the solution-phase ionization of weak acid residues; e.g., reducing opposite charging. Here the abilities of analogous compounds to increase or decrease charging in positive ion mode ESI are predicted from experimentally measured basicities. Consistently, amides, nitriles, and pyrazoles more basic than water reduced the average charge of protein analytes electrosprayed from denaturing solutions, while analogues less basic than water increased the average charge, establishing the veracity of solvent leveling as a supercharging mechanism. In other words, reducing the charge departing on solvent leaves more charge for the protein analyte.

Journal Article↗

PubMed2026

A two-factor authentication mechanism licenses pilins for pilus assembly in gram-positive bacteria.

Gram-positive bacteria display virulence-associated pili that facilitate adhesion and biofilm formation. These pili are covalently polymerized by class C sortase enzymes, which selectively recognize their cognate pilin substrates amid numerous cell wall sorting signal (CWSS)-bearing proteins. The molecular basis for this stringent substrate specificity has remained unclear. Here, we develop a rapid, quantitative fluorescence-activated cell sorting assay to monitor pilus assembly in Corynebacterium diphtheriae, enabling high-throughput analysis of SpaA pilin and SrtA sortase variants. Using this platform, together with molecular modeling and dynamics simulations, we show that SrtA engages nearly the entire SpaA CWSS to form a membrane-embedded complex that incorporates not only the LPXTG motif but also its connector and transmembrane helix elements. Formation of this interface displaces an inhibitory active-site lid and activates the enzyme to load the pilin substrate. Systematic CWSS swapping experiments and deep mutational scanning further support this model, demonstrating that noncognate pilins are excluded because they fail to form the required interface. Conversely, SrtA variants with an artificially unlatched lid bypass the need for this interface, indicating that membrane-driven complex formation is important for substrate licensing. Together, these findings define a "two-factor authentication" mechanism for pilus assembly in gram-positive bacteria: class C sortases first verify pilin identity by forming a membrane-embedded interface that activates the enzyme, then they recognize the LPXTG motif to initiate loading and crosslinking. This work provides a unified molecular framework for selective pilin incorporation in gram-positive bacteria and identifies potential vulnerabilities in the licensing machinery that may be exploited therapeutically.

Fimbriae, Bacterial↗

PubMed2026

Cryo-EM provides insight into how the Staphylococcus aureus IsdH receptor removes hemin from the hemoglobin:haptoglobin complex.

Staphylococcus aureus extracts hemin from human hemoglobin (Hb) to overcome host-imposed iron limitation. How it recovers Hb-bound hemin from the hemoglobin:haptoglobin (Hb:Hp) complex, the major circulating form of Hb outside red blood cells, remains unclear. Here we use cryo-electron microscopy, biophysical measurements, and solution kinetics to define how the S. aureus IsdH surface receptor extracts hemin from Hb:Hp. A 3.1 Å cryo-EM structure of Hb:Hp bound by full-length IsdH reveals that its N-terminal NEAT domain (N1) anchors it to αHb, whereas its downstream N2N3 extraction unit engages βHb to remove its hemin. The receptor engages Hb:Hp differently than isolated Hb, because N-linked glycans on haptoglobin bias the extraction unit toward βHb, sterically occluding its access to αHb while still permitting engagement by N1. Kinetic assays show that IsdH actively accelerates hemin release from Hb:Hp. Three-dimensional variability analysis indicates that this likely occurs via a dynamic interface in which receptor motions reposition the extraction unit relative to βHb, collectively supporting a model in which IsdH transiently perturbs the F-helix to promote hemin extraction. Alignment of that model with a previously determined CD163:Hb:Hp structure shows how IsdH may disrupt Hb:Hp recognition by macrophage and monocyte CD163 receptors, helping to explain how it may hinder clearance of Hb:Hp from circulation. In aggregate, these results help define the structural basis for hemin extraction from Hb:Hp and how IsdH may subvert receptor-mediated clearance of the Hb:Hp complex.

Journal Article↗

PubMed2025

Retention and Rearrangement of Membrane Protein Complexes' Higher Order Structure by Collisionally Activated Dissociation- and Electron Capture Dissociation-Mass Spectrometry.

Membrane proteins (MPs) challenge biophysical and structural biology methods. Native mass spectrometry (MS) has emerged as a powerful tool to study MP structures and their modulation by lipids. We examine the pathways by which three MPs, tetrameric Aquaporin Z (AqpZ), trimeric ammonium transporter (AmtB), and pentameric mechanosensitive channel of large conductance (MscL) decompose following collisionally activated dissociation (CAD) or electron capture dissociation (ECD) in native top-down MS. MPs subjected to CAD typically decompose along well-characterized pathways releasing highly charged monomers and low-charge state complementary subunits, but a lesser pathway accessed by low pressure collisions cleaves the backbone into fragments covering much of the sequence. Collisions also rearrange structures, e.g., AmtB subunits rearrange to form novel interactions and/or salt bridges that surprisingly retain a formerly surface-exposed segment despite ejecting the binding interface. MscL dissociation pathways depend on precursor charge-state, a behavior observed in a few soluble complexes, yet unaddressed mechanistically. Salt bridges in low charge state complexes stabilize subunits from ejection while facilitating only smaller, local rearrangements that release covalently cleaved products from transmembrane regions. With fewer opposite charges, on average, higher charge state molecules can rearrange intersubunit salt bridges on the experimental time scale to partition charge asymmetrically and free a subunit. ECD with supplemental activation can retain higher order structures of proteins and inform about the strongly interacting regions that preclude product ion release. With extensive regions lacking ionizable residues, MPs enable key interactions that guide the structure and dynamics of gas phase protein assemblies to be probed.

Mass Spectrometry↗

PubMed2025

Leveraging bioorthogonal conjugation for alpha synuclein fibril surveillance.

Alpha synuclein (α-syn) amyloid fibrils are associated with various neurodegenerative diseases. To better understand the molecular and cellular basis for α-syn fibril persistence and spread, we implemented a fluorophore labeling strategy to surveil pre-formed α-syn fibrils in solution and in cells. We leveraged amber codon mediated incorporation of a tetrazine-based artificial amino acid (TetV2.0) to install a cyclooctene-conjugated Janeliaflour, JF549, at four sites on human α-syn: residues 4, 60, 96 and 136. Fast coupling occurred under mild buffer conditions and in the presence of the disease-associated cofactor and cytotoxic lipid, psychosine. Labeled fibrils retained their polymorphic features, seeded the growth of new fibrils in vitro, and induced the seeding of positive puncta in α-syn FRET biosensor HEK293T cells. This allowed simultaneous tracking of exogenous and endogenous α-syn aggregates in biosensor cells, and their localization within the cells. In doing so, our approach facilitates more detailed mechanistic investigation of α-syn aggregates.

Synuclein↗