Bacteriorhodopsin: Structural Insights Revealed Using X-Ray Lasers and Synchrotron Radiation.

Directional transport of protons across an energy transducing membrane-proton pumping-is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-trans retinal chromophore being photoisomerized to a 13-cis conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive.

Mfd Dynamically Regulates Transcription via a Release and Catch-Up Mechanism.

The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd's ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive. Here, using a novel real-time translocase assay, we unexpectedly discovered that Mfd translocates autonomously on DNA. The speed and processivity of Mfd dictate a "release and catch-up" mechanism to efficiently patrol DNA for frequently stalled RNAPs. Furthermore, we showed that Mfd prevents RNAP backtracking or rescues a severely backtracked RNAP, allowing RNAP to overcome stronger obstacles. However, if an obstacle's resistance is excessive, Mfd dissociates the RNAP, clearing the DNA for other processes. These findings demonstrate a remarkably delicate coordination between Mfd and RNAP, allowing efficient targeting and recycling of Mfd and expedient conflict resolution.

XRCC1 prevents toxic PARP1 trapping during DNA base excision repair.

Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase ß and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase ß and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1-/- cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.

The Oncogenic Helicase ALC1 Regulates PARP Inhibitor Potency by Trapping PARP2 at DNA Breaks.

The anti-tumor potency of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) has been linked to trapping of PARP1 on damaged chromatin. However, little is known about their impact on PARP2, an isoform with overlapping functions at DNA lesions. Whether the release of PARP1/2 from DNA lesions is actively catalyzed by molecular machines is also not known. We found that PARPis robustly trap PARP2 and that the helicase ALC1 (CHD1L) is strictly required for PARP2 release. Catalytic inactivation of ALC1 quantitatively traps PARP2 but not PARP1. ALC1 manipulation impacts the response to single-strand DNA breaks through PARP2 trapping, potentiates PARPi-induced cancer cell killing, and mediates synthetic lethality upon BRCA deficiency. The chromatin remodeler ALC1 actively drives PARP2 turnover from DNA lesions, and PARP2 contributes to the cellular responses of PARPi. This suggests that disrupting the ATP-fueled remodeling forces of ALC1 might enable therapies that selectively target the DNA repair functions of PARPs in cancer.

The complex universe of inactive PARP1.

Poly(ADP-ribose) polymerase 1 (PARP1) is a crucial member of the PARP family, which modifies targets through ADP-ribosylation and plays key roles in a variety of biological processes. PARP inhibitors (PARPis) hinder ADP-ribosylation and lead to the retention of PARP1 at the DNA lesion (also known as trapping), which underlies their toxicity. However, inhibitors and mutations that make PARP1 inactive do not necessarily correlate with trapping potency, challenging the current understanding of inactivation-caused trapping. Recent studies on mouse models indicate that both trapping and non-trapping inactivating mutations of PARP1 lead to embryonic lethality, suggesting the unexpected toxicity of the current inhibition strategy. The allosteric model, complicated automodification, and various biological functions of PARP1 all contribute to the complexity of PARP1 inactivation.

Discovering optimal kinetic pathways for self-assembly using automatic differentiation.

Macromolecular complexes are often composed of diverse subunits. The self-assembly of these subunits is inherently nonequilibrium and must avoid kinetic traps to achieve high yield over feasible timescales. We show how the kinetics of self-assembly benefits from diversity in subunits because it generates an expansive parameter space that naturally improves the "expressivity" of self-assembly, much like a deeper neural network. By using automatic differentiation algorithms commonly used in deep learning, we searched the parameter spaces of mass-action kinetic models to identify classes of kinetic protocols that mimic biological solutions for productive self-assembly. Our results reveal how high-yield complexes that easily become kinetically trapped in incomplete intermediates can instead be steered by internal design of rate-constants or external and active control of subunits to efficiently assemble. Internal design of a hierarchy of subunit binding rates generates self-assembly that can robustly avoid kinetic traps for all concentrations and energetics, but it places strict constraints on selection of relative rates. External control via subunit titration is more versatile, avoiding kinetic traps for any system without requiring molecular engineering of binding rates, albeit less efficiently and robustly. We derive theoretical expressions for the timescales of kinetic traps, and we demonstrate our optimization method applies not just for design but inference, extracting intersubunit binding rates from observations of yield-vs.-time for a heterotetramer. Overall, we identify optimal kinetic protocols for self-assembly as a powerful mechanism to achieve efficient and high-yield assembly in synthetic systems whether robustness or ease of "designability" is preferred.

Spatiotemporal formation of a single liquid-like condensate and amyloid fibrils of α-synuclein by optical trapping at solution surface.

Liquid-like protein condensates have recently attracted much attention due to their critical roles in biological phenomena. They typically show high fluidity and reversibility for exhibiting biological functions, while occasionally serving as sites for the formation of amyloid fibrils. To comprehend the properties of protein condensates that underlie biological function and pathogenesis, it is crucial to study them at the single-condensate level; however, this is currently challenging due to a lack of applicable methods. Here, we demonstrate that optical trapping is capable of inducing the formation of a single liquid-like condensate of α-synuclein in a spatiotemporally controlled manner. The irradiation of tightly focused near-infrared laser at an air/solution interface formed a condensate under conditions coexisting with polyethylene glycol. The fluorescent dye-labeled imaging showed that the optically induced condensate has a gradient of protein concentration from the center to the edge, suggesting that it is fabricated through optical pumping-up of the α-synuclein clusters and the expansion along the interface. Furthermore, Raman spectroscopy and thioflavin T fluorescence analysis revealed that continuous laser irradiation induces structural transition of protein molecules inside the condensate to ß-sheet rich structure, ultimately leading to the condensate deformation and furthermore, the formation of amyloid fibrils. These observations indicate that optical trapping is a powerful technique for examining the microscopic mechanisms of condensate appearance and growth, and furthermore, subsequent aging leading to amyloid fibril formation.

Conservation of kinetic stability, but not the unfolding mechanism, between human transthyretin and a transthyretin-related enzyme.

Kinetic stability is thought to be an attribute of proteins that require a long lifetime, such as the transporter of thyroxine and holo retinol-binding protein or transthyretin (TTR) functioning in the bloodstream, cerebrospinal fluid, and vitreous humor. TTR evolved from ancestral enzymes known as TTR-related proteins (TRPs). Here, we develop a rate-expansion approach that allows unfolding rates to be measured directly at low denaturant concentration, revealing that kinetic stability exists in the Escherichia coli TRP (EcTRP), even though the enzyme structure is more energetically frustrated and has a more mutation-sensitive folding mechanism than human TTR. Thus, the ancient tetrameric enzyme may already have been poised to mutate into a kinetically stable human transporter. An extensive mutational study that exchanges residues at key sites within the TTR and EcTRP dimer-dimer interface shows that tyrosine 111, replaced by a threonine in TTR, is the gatekeeper of frustration in EcTRP because it is critical for function. Frustration, virtually absent in TTR, occurs at multiple sites in EcTRP and even cooperatively for certain pairs of mutations. We present evidence that evolution at the C terminus of TTR was a compensatory event to maintain the preexisting kinetic stability while reducing frustration and sensitivity to mutation. We propose an "overcompensation" pathway from EcTRPs to functional hybrids to modern TTRs that is consistent with the biophysics discussed here. An alternative plausible pathway is also presented.

Lipid-derived electrophiles inhibit the function of membrane channels during ferroptosis.

The therapeutic targeting of ferroptosis requires full understanding of the molecular mechanism of this regulated cell death pathway. While lipid-derived electrophiles (LDEs), including 4-hydroxy-2-nonenal (4-HNE), are important biomarkers of ferroptosis, a functional role for these highly reactive species in ferroptotic cell death execution has not been established. Here, through mechanistic characterization of LDE-detoxification impairment, we demonstrate that LDEs mediate altered protein function during ferroptosis. Applying live cell fluorescence imaging, we first identified that export of glutathione-LDE-adducts through multidrug resistance-associated protein (MRP) channels is inhibited following exposure to a panel of ferroptosis inducers (FINs) with different modes of action (type I-IV FINs erastin, RSL3, FIN56, and FINO2). This channel inhibition was recreated by both initiation of lipid peroxidation and treatment with 4-HNE. Importantly, treatment with radical-trapping antioxidants prevented impaired LDE-adduct export when working with both FINs and lipid peroxidation initiators but not 4-HNE, pinpointing LDEs as the cause of this inhibited MRP activity observed during ferroptosis. Our findings, when combined with reports of widespread LDE alkylation of key proteins following ferroptosis induction, including MRP1, set a precedent for LDEs as critical mediators of ferroptotic cell damage. Lipid hydroperoxide breakdown to form truncated phospholipids and LDEs may fully explain membrane permeabilization and modified protein function downstream of lipid peroxidation, offering a unified explanation of the molecular cell death mechanism of ferroptosis.

Molecular mechanism of the metal-independent production of hydroxyl radicals by thiourea dioxide and H2O2.

It is well-known that highly reactive hydroxyl radicals (HO•) can be produced by the classic Fenton system and our recently discovered haloquinone/H2O2 system, but rarely from thiol-derivatives. Here, we found, unexpectedly, that HO• can be generated from H2O2 and thiourea dioxide (TUO2), a widely used and environmentally friendly bleaching agent. A carbon-centered radical and sulfite were detected and identified as the transient intermediates, and urea and sulfate as the final products, with the complementary application of electron spin-trapping, oxygen-18 isotope labeling coupled with HPLC/MS analysis. Density functional theory calculations were conducted to further elucidate the detailed pathways for HO• production. Taken together, we proposed that the molecular mechanism for HO• generation by TUO2/H2O2: TUO2 tautomerizes from sulfinic acid into ketone isomer (TUO2-K) through proton transfer, then a nucleophilic addition of H2O2 on the S atom of TUO2-K, forming a S-hydroperoxide intermediate TUO2-OOH, which dissociates homolytically to produce HO•. Our findings represent the first experimental and computational study on an unprecedented new molecular mechanism of HO• production from simple thiol-derived sulfinic acids, which may have broad chemical, environmental, and biomedical significance for future research on the application of the well-known bleaching agent and its analogs.

Utilizing the oxygen-atom trapping effect of Co3O4 with oxygen vacancies to promote chlorite activation for water decontamination.

Heterogeneous high-valent cobalt-oxo [≡Co(IV)=O] is a widely focused reactive species in oxidant activation; however, the relationship between the catalyst interfacial defects and ≡Co(IV)=O formation remains poorly understood. Herein, photoexcited oxygen vacancies (OVs) were introduced into Co3O4 (OV-Co3O4) by a UV-induced modification method to facilitate chlorite (ClO2-) activation. Density functional theory calculations indicate that OVs result in low-coordinated Co atom, which can directionally anchor chlorite under the oxygen-atom trapping effect. Chlorite first undergoes homolytic O-Cl cleavage and transfers the dissociated O atom to the low-coordinated Co atom to form reactive ≡Co(IV)=O with a higher spin state. The reactive ≡Co(IV)=O rapidly extracts one electron from ClO2- to form chlorine dioxide (ClO2), accompanied by the Co atom returning a lower spin state. As a result of the oxygen-atom trapping effect, the OV-Co3O4/chlorite system achieved a 3.5 times higher efficiency of sulfamethoxazole degradation (~0.1331 min-1) than the pristine Co3O4/chlorite system. Besides, the refiled OVs can be easily restored by re-exposure to UV light, indicating the sustainability of the oxygen atom trap. The OV-Co3O4 was further fabricated on a polyacrylonitrile membrane for back-end water purification, achieving continuous flow degradation of pollutants with low cobalt leakage. This work presents an enhancement strategy for constructing OV as an oxygen-atom trapping site in heterogeneous advanced oxidation processes and provides insight into modulating the formation of ≡Co(IV)=O via defect engineering.

Post-Transcriptional Regulation of Rab7a in Lysosomal Positioning and Drug Resistance in Nutrient-Limited Cancer Cells.

Limited nutrient availability in the tumor microenvironment can cause the rewiring of signaling and metabolic networks to confer cancer cells with survival advantages. We show here that the limitation of glucose, glutamine and serum from the culture medium resulted in the survival of a population of cancer cells with high viability and capacity to form tumors in vivo. These cells also displayed a remarkable increase in the abundance and size of lysosomes. Moreover, lysosomes were located mainly in the perinuclear region in nutrient-limited cells; this translocation was mediated by a rapid post-transcriptional increase in the key endolysosomal trafficking protein Rab7a. The acidic lysosomes in nutrient-limited cells could trap weakly basic drugs such as doxorubicin, mediating resistance of the cells to the drug, which could be partially reversed with the lysosomal inhibitor bafilomycin A1. An in vivo chorioallantoic membrane (CAM) assay indicated a remarkable decrease in microtumor volume when nutrient-limited cells were treated with 5-Fluorouracil (5-FU) and bafilomycin A1 compared to cells treated with either agent alone. Overall, our data indicate the activation of complementary pathways with nutrient limitation that can enable cancer cells to survive, proliferate and acquire drug resistance.

Nicotine inhalation and metabolism triggers AOX-mediated superoxide generation with oxidative lung injury.

With the increasing use of vaping devices that deliver high levels of nicotine (NIC) to the lungs, sporadic lung injury has been observed. Commercial vaping solutions can contain high NIC concentrations of 150 mM or more. With high NIC levels, its metabolic products may induce toxicity. NIC is primarily metabolized to form NIC iminium (NICI) which is further metabolized by aldehyde oxidase (AOX) to cotinine. We determine that NICI in the presence of AOX is a potent trigger of superoxide generation. NICI stimulated superoxide generation from AOX with Km = 2.7 µM and Vmax = 794 nmol/min/mg measured by cytochrome-c reduction. EPR spin-trapping confirmed that NICI in the presence of AOX is a potent source of superoxide. AOX is expressed in the lungs and chronic e-cigarette exposure in mice greatly increased AOX expression. NICI or NIC stimulated superoxide production in the lungs of control mice with an even greater increase after chronic e-cigarette exposure. This superoxide production was quenched by AOX inhibition. Furthermore, e-cigarette-mediated NIC delivery triggered oxidative lung damage that was blocked by AOX inhibition. Thus, NIC metabolism triggers AOX-mediated superoxide generation that can cause lung injury. Therefore, high uncontrolled levels of NIC inhalation, as occur with e-cigarette use, can induce oxidative lung damage.

Rational design of eukaryotic riboswitches that up-regulate IRES-mediated translation initiation with high switching efficiency through a kinetic trapping mechanism in vitro.

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

Steric trapping strategy for studying the folding of helical membrane proteins.

Elucidating the folding energy landscape of membrane proteins is essential to the understanding of the proteins' stabilizing forces, folding mechanisms, biogenesis, and quality control. This is not a trivial task because the reversible control of folding is inherently difficult in a lipid bilayer environment. Recently, novel methods have been developed, each of which has a unique strength in investigating specific aspects of membrane protein folding. Among such methods, steric trapping is a versatile strategy allowing a reversible control of membrane protein folding with minimal perturbation of native protein-water and protein-lipid interactions. In a nutshell, steric trapping exploits the coupling of spontaneous denaturation of a doubly biotinylated protein to the simultaneous binding of bulky monovalent streptavidin molecules. This strategy has been evolved to investigate key elements of membrane protein folding such as thermodynamic stability, spontaneous denaturation rates, conformational features of the denatured states, and cooperativity of stabilizing interactions. In this review, we describe the critical methodological advancement, limitation, and outlook of the steric trapping strategy.

A Novel Air Trapping Segment Score Identifies Opposing Effects of Obesity and Eosinophilia on Air Trapping in Asthma.

Rationale: Density thresholds in computed tomography (CT) lung scans quantify air trapping (AT) at the whole-lung level but are not informative for AT in specific bronchopulmonary segments. Objectives: To apply a segment-based measure of AT in asthma to investigate the clinical determinants of AT in asthma. Methods: In each of 19 bronchopulmonary segments in CT lung scans from 199 patients with asthma, AT was categorized as present if lung attenuation was less than -856 Hounsfield units at expiration in ⩾15% of the lung area. The resulting AT segment score (0-19) was related to patient outcomes. Measurements and Main Results: AT varied at the lung segment level and tended to persist at the patient and lung segment levels over 3 years. Patients with widespread AT (⩾10 segments) had more severe asthma (P < 0.05). The mean (±SD) AT segment score in patients with a body mass index ⩾30 kg/m2 was lower than in patients with a body mass index <30 kg/m2 (3.5 ± 4.6 vs. 5.5 ± 6.3; P = 0.008), and the frequency of AT in lower lobe segments in obese patients was less than in upper and middle lobe segments (35% vs. 46%; P = 0.001). The AT segment score in patients with sputum eosinophils ⩾2% was higher than in patients without sputum eosinophilia (7.0 ± 6.1 vs. 3.3 ± 4.9; P < 0.0001). Lung segments with AT more frequently had airway mucus plugging than lung segments without AT (48% vs. 18%; P ⩽ 0.0001). Conclusions: In patients with asthma, air trapping is more severe in those with airway eosinophilia and mucus plugging, whereas those who are obese have less severe trapping because their lower lobe segments are spared.

Assignment of structural transitions during mechanical unwrapping of nucleosomes and their disassembly products.

Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetric, occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection, we show that the low-force transition corresponds to the undoing of the outer wrap of one side of the nucleosome (∼27 bp), a process that can occur either cooperatively or noncooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ∼76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition and another that coincides but does not contribute to the high-force transition.

Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein.

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

In situ optical spectroscopy of crystallization: One crystal nucleation at a time.

While crystallization is a ubiquitous and an important process, the microscopic picture of crystal nucleation is yet to be established. Recent studies suggest that the nucleation process can be more complex than the view offered by the classical nucleation theory. Here, we implement single crystal nucleation spectroscopy (SCNS) by combining Raman microspectroscopy and optical trapping induced crystallization to spectroscopically investigate one crystal nucleation at a time. Raman spectral evolution during a single glycine crystal nucleation from water, measured by SCNS and analyzed by a nonsupervised spectral decomposition technique, uncovered the Raman spectrum of prenucleation aggregates and their critical role as an intermediate species in the dynamics. The agreement between the spectral feature of prenucleation aggregates and our simulation suggests that their structural order emerges through the dynamic formation of linear hydrogen-bonded networks. The present work provides a strong impetus for accelerating the investigation of crystal nucleation by optical spectroscopy.

One-Dimensional MoS2 Nanoscrolls as Miniaturized Memories.

Dimensionality of materials is closely related to their physical properties. For two-dimensional (2D) semiconductors such as monolayer molybdenum disulfide (MoS2), converting them from 2D nanosheets to one-dimensional (1D) nanoscrolls could contribute to remarkable electronic and optoelectronic properties, yet the rolling-up process still lacks sufficient controllability, which limits the development of their device applications. Herein we report a modified solvent evaporation-induced rolling process that halts at intermediate states and achieve MoS2 nanoscrolls with high yield and decent axial uniformity. The accordingly fabricated nanoscroll memories exhibit an on/off ratio of ∼104 and a retention time exceeding 103 s and can realize multilevel storage with pulsed gate voltages. Such open-end, high-curvature, and hollow 1D nanostructures provide new possibilities to manipulate the hysteresis windows and, consequently, the charge storage characteristics of nanoscale field-effect transistors, thereby holding great promise for the development of miniaturized memories.