Yun-Wei Chiang

Although biological membranes exhibit complex and dynamic organization, the mechanistic role of molecular crowding in governing lateral membrane heterogeneity remains poorly characterized experimentally. Here, we provide direct experimental visualization showing that crowding‐induced condensation of membrane‐anchored macromolecules above the bilayer interface reorganizes membrane dynamics and gives rise to spatially heterogeneous lipid mobility. Both PEGylated lipids and membrane‐anchored proteins undergo surface‐density‐driven condensation on supported lipid bilayers (SLBs), forming immobile regions that constrain lipid diffusion. Notably, the condensation threshold varies inversely with poly(ethylene glycol) chain length and surface density, defining a quantitative relationship between molecular size and crowding strength. To directly visualize these crowding‐induced structures, we employed a nanodisc‐mediated lipid delivery (“nanodelivery”) approach, revealing a clear correspondence between condensed crowder regions and diffusion barriers reminiscent of the picket‐fence model of live‐cell membranes. Similar behavior in protein‐crowded SLBs demonstrates the generality of this crowding‐driven mechanism. Using minimal two‐layer and three‐layer reconstituted architectures, we further demonstrate that surface density and macromolecular crowding above the membrane function as tunable control parameters that drive phase transitions within membrane‐tethered macromolecular layers, independent of anchoring chemistry, while the underlying lipid bilayer remains laterally continuous. Extension of this framework to living cells reveals analogous suppression of membrane mobility under high surface‐density conditions, indicating that this crowding‐induced reorganization is not restricted to SLBs model systems. Together, these findings establish surface‐density‐driven phase transitions of membrane‐anchored macromolecules as a general physical mechanism underlying lateral membrane inhomogeneity and position nanodelivery as a versatile approach for interrogating membrane organization across synthetic and biological systems.

Proton-coupled ion transport is a fundamental chemical process underlying membrane physiology, yet how local electrostatics are transduced into gated Ca ²⁺ permeation remains poorly defined. Here, we combine single-channel planar bilayer electrophysiology, nanodisc-based double electron–electron resonance spectroscopy, atomistic modeling, and a nanodisc nano-delivery strategy that enables direct functional insertion of purified membrane proteins into live mammalian cells. Applying this integrated toolkit to the bacterial transmembrane Bax-inhibitor-1–containing motif prototype Bs YetJ, we resolve a hierarchical electrostatic gating mechanism governed by two salt bridges with distinct physical roles. A periplasmic E49–R205 interaction functions as a proton-sensitive latch that drives transmembrane helix 2 displacement and controls opening probability, while a cytoplasmic E182–R15 pair operates as a local electrostatic determinant of Ca ²⁺ self-block that tunes conductance and selectivity without large-scale conformational change. Quantitative separation of these effects reveals how protonation reshapes the energy landscape of ion permeation. Live-cell Ca ²⁺ imaging following nano-delivery recapitulates this gating logic in a cellular membrane setting. Together, this work establishes dual salt-bridge electrostatics as a chemical principle for graded Ca ²⁺ leak and introduces nano-delivery as a powerful platform for connecting molecular electrostatics to cellular ion transport.

Nonheme diiron enzymes catalyze a wide range of biologically essential redox transformations. Despite extensive study, how the surrounding protein matrix coordinates electron transfer, proton delivery, and solvent access and whether mechanistic insights derived in vitro reflect enzyme function in living systems remain incompletely understood. Here, we investigate the bacterial enzyme ScdA, a nonheme diiron nitrite reductase that converts nitrite to nitric oxide (NO), to define how first- and second-sphere interactions regulate catalysis. Using site-directed mutagenesis, steady-state kinetics, EPR spectroscopy, solvent kinetic isotope effect analysis, molecular dynamics simulations, and cell-based spin trapping, we identify distinct functional contributions of residues surrounding the diiron center. First-sphere ligands ensure cofactor assembly and redox integrity, whereas second-sphere residues modulate turnover by controlling hydrogen-bond networks and solvent accessibility near the catalytic core. Structural and kinetic analyses reveal a solvent-accessible pathway whose gating properties tune hydration dynamics and influence the rate-limiting step. Importantly, cell-based EPR detection of NO demonstrates that the same structural determinants governing catalytic efficiency in vitro also operate under cellular conditions. Together, these results establish controlled hydration as a general design principle in nonheme diiron enzymes.

Singlet fission (SF) offers a promising avenue for quantum information science, as it generates spin-entangled triplet pairs with quintet character (⁵TT) upon photoexcitation, enabling access to multilevel spin qubit states beyond the traditional two-level systems. However, the ⁵TT state often decays via several pathways: (1) dissociation into isolated triplets; (2) triplet-triplet annihilation back into the singlet manifold; or (3) spin conversion to lower-multiplicity triplet pair states. These competing relaxation channels pose a major challenge for stabilizing ⁵TT. Here, we introduce a novel molecular design that prolongs ⁵TT lifetime by anchoring two pentacene chromophores to the same carbon (C9) position of a fluorene bridge, yielding FlePc2 and FlePhPc2. This single-point attachment enforces a near-parallel intramolecular geometry, promoting strong through-space spin interactions that hinder dissociation. Field-swept electron spin echo (FS-ESE) measurements reveal dominant ⁵TT signals, indicative of suppressed relaxation pathways. Theoretical calculations predict a substantial binding energy for the reported dimers, accompanied by significant spin density delocalization across both pentacenes, thereby rationalizing ⁵TT stabilization. These findings establish a molecular design principle for kinetically trapping high-spin multiexciton states, paving the way for spin-based quantum technologies.

Pathogenic Staphylococcus aureus endures bursts of host-derived reactive nitrogen species, yet the molecular defenses that enable this resilience have remained unclear. We now show that the previously enigmatic di-iron enzyme ScdA functions as a nitrite reductase, converting nitrite to nitric oxide (NO), and we elucidate the structural elements that support this activity. Using an integrative toolkit─X-ray crystallography, solution NMR, AlphaFold modeling, and pulsed EPR/DEER─we solved the full-length homodimeric structure of ScdA and identified a robust di-iron center that forms stable iron-nitrosyl intermediates. Targeted mutagenesis reveals that redox-active cysteines and dimerization state tune catalytic output, whereas steady-state kinetics confirm efficient nitrite-to-NO turnover. In vivo, ScdA overexpression in Escherichia coli suppresses growth under nitrite-rich conditions, highlighting the cytotoxic potency of the NO it generates. By coupling structure to function, our work clarifies S. aureus strategies for managing nitrosylative stress and points to ScdA as a potential vulnerability in antibiotic-resistant pathogens.

The canonical LQTD motif (residues 56-59) has long been regarded as the sole "address tag" that directs Caspase-8 to cleave the pro-apoptotic protein Bid at D59. Here we overturn this view, showing that Bid's previously uncharted 42-residue "disordered" loop furnishes a second layer of control that can either accelerate or block cleavage. Alanine scanning, MD simulations, DEER spectroscopy, and AlphaFold reveal two physical modules in this loop: an electrostatic clamp formed by E53-D54-E55 that secures the substrate upstream of LQTD, and an entropic flexibility switch at S61-Q62 that modulates catalysis. Phosphorylation at S61 locks the switch and halts proteolysis, whereas a double-alanine substitution (S61A-Q62A) loosens intraloop hydrogen bonding and boosts cleavage by ∼50%. Loop charge, dynamics, and post-translational modification thus cooperate to set Caspase-8 specificity, establishing dynamic loops as tunable physical targets for chemical control of apoptotic signaling.

Bacterial outer membrane vesicles (OMVs) have emerged as promising vehicles for anticancer drug delivery due to their inherent tumor tropism, immune-stimulatory properties, and potential for functionalization with therapeutic proteins. Despite their advantages, the high lipopolysaccharide (LPS) endotoxin content in the OMVs raises significant safety and regulatory challenges. In this work, we produce LPS-attenuated and LPS-free OMVs and systematically assess the effects of LPS modification on OMVs' physicochemical characteristics, membrane protein content, immune-stimulatory capacity, tolerability, and anticancer efficacy. Our findings reveal that LPS removal increased the maximal tolerated dose of the OMVs by over 25-fold. When adjusted for comparable safety profiles, LPS-free OMVs exhibit superior anticancer effects compared with wild-type OMVs. Mechanistic investigations indicate that the LPS removal obviates immune cell death caused by LPS and reduces the negatory effects of wild type of OMVs on tumor immune cell infiltrates. We further show the functionality of the LPS-free OMV through the incorporation of an IL-2 variant protein (Neo-2/15). This functionalization augments OMV's ability of the OMV to inhibit tumor growth and promote lymphocyte infiltration into the tumor microenvironment. This study presents a safe and functionalizable OMV with improved translational prospect.

The diiron‐containing YtfE protein in Escherichia coli is pivotal in counteracting nitrosative stress, a critical barrier to bacterial viability. This study delves into the biochemical complexity governing YtfE's conversion of nitric oxide (NO) to nitrous oxide, a key process for alleviating nitrosative stress. Through site‐directed mutagenesis, we explored YtfE's molecular structure, with a particular focus on two internal transport tunnels important for its activity. Our findings illuminate Tunnel 1 as the primary conduit for substrate transport, regulated by conformational shifts within the N‐terminal domain that enable substrate access to the diiron core in the C‐terminal domain. Tunnel 2 emerges as a secondary, supportive route, activated when Tunnel 1 is compromised. This result challenges a previous model of distinct tunnels for substrate entry and product exit, suggesting both tunnels are capable of transporting substrates and products. Our engineering efforts enhanced the role of Tunnel 2, enabling a synergistic operation with Tunnel 1 and tripling YtfE's enzymatic activity compared to its wild‐type form. This research not only deepens our understanding of YtfE's regulatory mechanism for NO reduction but also introduces a strategy to amplify its enzymatic efficiency. The outcomes offer new ravenues for modulating bacterial stress responses.

Membrane proteins are integral to numerous cellular processes, yet their conformational dynamics in native environments remains difficult to study. This study introduces a nanodelivery method using nanodiscs to transport spin-labeled membrane proteins into the membranes of living cells, enabling direct in-cell double electron-electron resonance (DEER) spectroscopy measurements. We investigated the membrane protein BsYetJ, incorporating spin labels at key positions to monitor conformational changes. Our findings demonstrate successful delivery and high-quality DEER data for BsYetJ in both Gram-negative E. coli and Gram-positive B. subtilis membranes. The delivered BsYetJ retains its ability to transport calcium ions. DEER analysis reveals distinct conformational states of BsYetJ in different membrane environments, highlighting the influence of lipid composition on the protein structure. This nanodelivery method overcomes traditional limitations, enabling the study of membrane proteins in more physiologically relevant conditions.