Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and how ESCRT-III ...shapes membranes is yet unclear. Human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and AFM. We show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes. Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes and reshape vesicles into helical "corkscrew-like" membrane tubes. Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and locally perpendicular to the tube axis, highlighting the mechanical stresses imposed by ESCRT-III. Our results underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes.
Biosorption is an ingenious technique that uses biological materials to acquire trace metal ions from wastewater. In the present study, the ability of
Colocasia esculenta
stem biomass was explored ...for the biosorption of toxic trace metals. The maximum removal was observed for arsenate (As
5+
) with 58.63%, followed by chromium (Cr
6+
) with 56.56%, and cadmium (Cd
2+
) with 41.2%. However, for copper (Cu
2+
), nickel (Ni
2+
), and zinc (Zn
2+
), low adsorption was observed. Batch sorption tests revealed that adsorbent dosage of 0.5g, 0.5g, and 0.3g; time of 10 h, 4 h, and 10 h; room temperature range of 25–30°C; pH range of 7.0–4.5; and initial concentration of 30 μg/L, 20 mg/L, and 30 mg/L were the optimum conditions for the removal of As
5+
, Cr
6+
, and Cd
2+
, respectively. Scanning electron microscope and energy-dispersive X-ray spectroscopy (SEM-EDX) analysis of
Colocasia esculenta
stem biomass before and after adsorption revealed that the trace metals successfully get adsorbed on the surface of the biosorbent. The equilibrium data fitted well with the adsorption isotherm model of Langmuir (for As
5+
, Cr
6+
, and Cd
2+
), Dubinin-Radushkevich (for As
5+
and Cr
6+
), and Flory-Huggins (for Cd
2+
), and the kinetic data of As
5+
, Cr
6+
, and Cd
2+
biosorption were best described by pseudo-second-order kinetic model. Thermodynamic studies revealed that the adsorption process for all concerned trace metals acts in a spontaneous manner and is endothermic in nature. Thus, the use of
Colocasia esculenta
stem biomass proved to be an efficient and economical alternative for the treatment of effluents contaminated with these trace metals.
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CEKLJ, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Direct visualization of pathways followed by single molecules while they spontaneously self-assemble into supramolecular biological machines may provide fundamental knowledge to guide molecular ...therapeutics and the bottom-up design of nanomaterials and nanodevices. Here, high-speed atomic force microscopy is used to visualize self-assembly of the bidimensional lattice of protein molecules that constitutes the framework of the mature human immunodeficiency virus capsid. By real-time imaging of the assembly reaction, individual transient intermediates and reaction pathways followed by single molecules could be revealed. As when assembling a jigsaw puzzle, the capsid protein lattice is randomly built. Lattice patches grow independently from separate nucleation events whereby individual molecules follow different paths. Protein subunits can be added individually, while others form oligomers before joining a lattice or are occasionally removed from the latter. Direct real-time imaging of supramolecular self-assembly has revealed a complex, chaotic process involving multiple routes followed by individual molecules that are inaccessible to bulk (averaging) techniques.
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IJS, KILJ, NUK, PNG, UL, UM
Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscope (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the ...unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment.
Abstract
Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance
1–3
. Teixobactin
4
represents a new class of antibiotics with a unique chemical scaffold and lack of ...detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan
5
. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin
4
. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
This investigation characterized the relationship between percentage occurrences of three size classes of Hilsa juveniles (3–5, 5.1–10, and 10.1–15 cm) and six biogeochemical parameters (water ...temperature, chlorophyll-
a
, turbidity, salinity, dissolved oxygen, and pH) in the lower part of the tidal riverine to the entire estuarine stretch of Hooghly. These relationships helped us determine the ranges of each biogeochemical parameter for different suitability criteria in three seasons, pre-monsoon (February–May), monsoon (June–September), and post-monsoon (October–January). Salinity had the highest weight (0.37–0.39) followed by chlorophyll-
a
(0.26–0.30) and turbidity (0.12–0.20). The geospatial data of the biogeochemical parameters were interpolated followed by reclassification with the suitability ranges, separately for each season. These reclassified data were integrated into GIS-based modeling by a multi-criteria decision-making technique (analytical hierarchy process) to generate the habitat suitability maps of juvenile Hilsa. The model-derived information was verified with the indigenous knowledge of the fishers regarding the suitable habitat of juvenile Hilsa by conducting group discussions at 13 locations along the entire study area. The degree of agreement/disagreement between the model and the field information was determined by measuring Kendall’s tau (0.81–0.96) and Kappa coefficients (0.77–0.86), which indicated a strong agreement. In total, 3.80%, 10.12%, and 31.08% of the total river-estuarine area considered for the present study were identified as highly suitable for juvenile Hilsa during pre-monsoon, monsoon, and post-monsoon seasons, respectively. This mapping can act as baseline information for the policymakers for sustainable Hilsa fishing keeping in view the livelihood of fishers.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The secondary active transporter CitS shuttles citrate across the cytoplasmic membrane of gram-negative bacteria by coupling substrate translocation to the transport of two Na
ions. Static crystal ...structures suggest an elevator type of transport mechanism with two states: up and down. However, no dynamic measurements have been performed to substantiate this assumption. Here, we use high-speed atomic force microscopy for real-time visualization of the transport cycle at the level of single transporters. Unexpectedly, instead of a bimodal height distribution for the up and down states, the experiments reveal movements between three distinguishable states, with protrusions of ∼0.5 nm, ∼1.0 nm, and ∼1.6 nm above the membrane, respectively. Furthermore, the real-time measurements show that the individual protomers of the CitS dimer move up and down independently. A three-state elevator model of independently operating protomers resembles the mechanism proposed for the aspartate transporter Glt
Since CitS and Glt
are structurally unrelated, we conclude that the three-state elevators have evolved independently.
Biogeochemical reduction and mobilization of sediment-bound arsenic (As) is the major concern for widespread groundwater As contamination in the middle Gangetic plains. The present work examines a ...microcosm based bio-stimulation study and substrate amendments over 45 days to analyze the bacterial community structure and distribution to indicate the possible in-situ bioremediation strategy in the area. Initially, Bacterial phyla
Proteobacteria
was predominantly present in all the samples, followed by
Actinobacteria
,
Bacteroidetes,
and
Firmicutes
whereas
Cyanobacteria
was noted as the minor group. In genus level,
Delftia
,
Acinetobacter, Lysobacter, Bacillus,
and
Pseudomonas
were the major groups of bacteria in the As-rich aquifer system, while
Planctomycetes
dominated the bio-stimulated samples, followed by a minute portion of
Proteobacteria
. Alpha diversity and Chaol curve further determined the species richness in the samples with an As tolerant capacity of 152.28 ppb. The presence of γ-
Proteobacteria
as the dominating member in high As-content water indicated their predominant role in As mobilization, whereas, dominance of
α-Proteobacterial
members in low As-content water indicated their involvement in As detoxification. The complete change in microbial community structure within the bio-stimulated conditions indicated the extensive role of arsenite-oxidizing microbial communities within different levels of As-contaminated areas in Bihar that will enlighten the significant role of these communities in As-biogeochemical cycle.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force ...microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes.
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