Additional file 2 of The small GTPase BcSec4 is involved in conidiophore development, membrane integrity, and autophagy in Botrytis cinerea

Additional file 2. Table S1. Conidiation-associated genes in B. cinere

Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States

Characterization of structural differences between coexisting conformational states of protein is difficult with conventional biophysical techniques. Hydrogen/deuterium exchange (HDX) coupled with top-down mass spectrometry (MS) allows different conformers to be deuterated to different extents and distinguished through gas-phase separation based on molecular weight distributions prior to determination of deuteration levels at local sites for each isolated conformer. However, application of this strategy to complex systems is hampered by the interference from conformers with only minor differences in overall deuteration levels. In this work, we performed differential HDX while the different conformers were separated according to their differing charge to size ratios in capillary electrophoresis. Mixtures of holo- and apo-myoglobin (Mb) and disulfide isomers of lysozyme (Lyz) were characterized in a conformer-specific fashion using this strategy, followed by conformation interrogation for the sequentially eluted 2H-labeled species in real-time using top-down MS. Under mildly denaturing conditions that minimize the charge difference, disulfide isomers of Lyz were differentially labeled with 2H during separation based on their disulfide-dependent sizes. The resulting differences in deuteration pattern between these isomers are in line with their difference in covalent structural constraints set by the disulfide patterns. Under physiologically relevant conditions, we identified the segments undergoing conformational changes of Mb in the absence of the heme group by comparing the deuteration patterns of holo- and apo-Mb

Additional file 1 of The small GTPase BcSec4 is involved in conidiophore development, membrane integrity, and autophagy in Botrytis cinerea

Additional file 1: Figure S1. BcSec4 is indispensable for vegetative growth of B. cinerea. a Colony morphology of B05.10, the ΔBcSEC4 mutants, and complemented strains grown on PDA plates for 4 days. b The ΔBcSEC4 mutant displayed enhanced hyphal branching compared to B05.10 after incubation on PDA plates for 3 days. c Morphology of hyphal tips of B05.10 and the ΔBcSEC4 mutant growing on PDA plates for 3 days. Figure S2. BcSec4 is involved in protein secretion. a The hypersensitive response in Nicotiana benthamiana elicited by the supernatants of B05.10 and the ΔBcSEC4 mutant. The supernatants were collected after incubation in YEPD liquid medium for 24 h. b SDS-PAGE analysis of extracellular proteins produced by B05.10 and the ΔBcSEC4 mutant. c Functional classification of extracellular proteins produced by B05.10 and the ΔBcSEC4 mutant. Figure S3. BcSec4 is required for full virulence of B. cinerea. Tomato (a) and mung bean (b) leaves were inoculated with mycelial plugs at 25 °C in the dark. Figure S4. Disruption of BcSEC4 did not impair infection cushion formation. a Infection cushion formation of B05.10 and the ΔBcSEC4 mutant on a glass surface at 24 hpi. b Quantification of infection cushion formation by B05.10 and the ΔBcSEC4 mutant. Figure S5. The expression levels of JA-related genes determined via RT-qPCR. Levels of transcripts were normalized against that of β-actin. Three biological replicates were performed independently. Data are represented as means ± standard deviations (SDs) from three independent experiment

Photo-responsive graphene oxide liquid crystal hybrid modified with imidazolium surfactant containing azobenzene

A novel photo-responsive imidazolium surfactant containing azobenzene mesogenic cores (CAzoCM) was designed and synthesised to modify graphene oxide, forming imidazolium surfactant-graphene oxide hybrid (CAzoCM-GO), focusing on the correlation between their mesomorphic behaviour, photo-responsive properties and their chemical structure. CAzoCM and CAzoCM-GO were characterised by FT−IR, Raman spectroscopy and XPS, respectively. The results showed that GO was partially reduced by CAzoCM due to the strong electron effect of the imidazole group. The liquid crystal properties of CAzoCM and CAzoCM-GO were investigated by DSC, POM and variable temperature XRD, respectively. CAzoCM exhibited a smectic A phase as well as the rare smectic C phase and the introduction of GO did not destroy the liquid crystal properties of CAzoCM. Furthermore, the photo-responsive behaviour of CAzoCM and CAzoCM-GO had also been studied by UV-vis spectrum. CAzoCM could be switched from trans to cis isomer in 270 s when it was irradiated under ultraviolet light and from cis to trans isomer in 660 s when it was irradiated under visible light. CAzoCM-GO exhibited more sensitive response behaviour, the photo-responsive time from trans to cis isomerisation became 110 s while the photo-responsive time from cis to trans isomerisation became 195 s.</p

Improving Accuracy in Mass Spectrometry-Based Mass Determination of Intact Heterogeneous Protein Utilizing the Universal Benefits of Charge Reduction and Alternative Gas-Phase Reactions

In mass analysis of proteins, mass spectrometry directly measures the mass to charge ratios of ionized proteins and promises higher accuracy than that of indirect approaches measuring other physicochemical properties, provided that the charge states of detected ions are determined. Accurate mass determination of heterogeneously glycosylated proteins is often hindered by unreliable charge determination due to the insufficient resolution of signals from different charge states and inconsistency among mass profiles of ions in individual charge states. Limited charge reduction of a subpopulation of proteoforms using electron transfer/capture reactions (ETnoD/ETnoD) solves this problem by narrowing the mass distribution of examined proteoforms and preserving the mass profile of the precursor charge state in the reduced charge states. However, the limited availability of ETnoD/ETnoD function in commercial instruments limits the application of this approach. Here, utilizing a range of charge-dependent and accuracy-affecting spectral features revealed by a systematic evaluation at levels of both the ensemble and subpopulation of proteoforms based on theoretical models and experiments, we developed a limited charge reduction workflow that enables using collision-induced dissociation and higher energy collisional dissociation, two widely available reactions, as alternatives to ETnoD/ETnoD while providing adequate accuracy. Alternatively, substituting proton transfer charge reduction for ETnoD/ETnoD provides higher accuracy of mass determination. Performing mass selection in a window-sliding manner improves the accuracy and allows profiling of the whole proteoform distribution. The proposed workflow may facilitate the development of universal characterization strategies for more complex and heterogeneous protein systems

sj-tex-1-smm-10.1177_09622802211046383 - Supplemental material for Modeling treatment effect modification in multidrug-resistant tuberculosis in an individual patientdata meta-analysis

Supplemental material, sj-tex-1-smm-10.1177_09622802211046383 for Modeling treatment effect modification in multidrug-resistant tuberculosis in an individual patientdata meta-analysis by Yan Liu, Mireille E Schnitzer, Guanbo Wang, Edward Kennedy, Piret Viiklepp, Mario H Vargas, Giovanni Sotgiu, Dick Menzies and Andrea Benedetti in Statistical Methods in Medical Research</p

Microfluidic Platform for Time-Resolved Characterization of Protein Higher-Order Structures and Dynamics Using Top-Down Mass Spectrometry

Characterization of protein higher-order structures and dynamics is essential for understanding the biological functions of proteins and revealing the underlying mechanisms. Top-down mass spectrometry (MS) accesses structural information at both the intact protein level and the peptide fragment level. Native top-down MS allows analysis of a protein complex’s architecture and subunits’ identity and modifications. Top-down hydrogen/deuterium exchange (HDX) MS offers high spatial resolution for conformational or binding interface analysis and enables conformer-specific characterization. A microfluidic chip can provide superior performance for front-end reactions useful for these MS workflows, such as flexibility in manipulating multiple reactant flows, integrating various functional modules, and automation. However, most microchip-MS devices are designed for bottom-up approaches or top-down proteomics. Here, we demonstrate a strategy for designing a microchip for top-down MS analysis of protein higher-order structures and dynamics. It is suitable for time-resolved native MS and HDX MS, with designs aiming for efficient ionization of intact protein complexes, flexible manipulation of multiple reactant flows, and precise control of reaction times over a broad range of flow rates on the submicroliter per minute scale. The performance of the prototype device is demonstrated by measurements of systems including monoclonal antibodies, antibody–antigen complexes, and coexisting protein conformers. This strategy may benefit elaborate structural analysis of biomacromolecules and inspire method development using the microchip-MS approach

Unraveling the Macromolecular Pathways of IgG Oligomerization and Complement Activation on Antigenic Surfaces

IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design

Unraveling the Macromolecular Pathways of IgG Oligomerization and Complement Activation on Antigenic Surfaces

IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design

Unraveling the Macromolecular Pathways of IgG Oligomerization and Complement Activation on Antigenic Surfaces

IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design