RESUMO
The ability to cope with and adapt to changes in the environment is essential for all organisms. Osmotic pressure is a universal threat when environmental changes result in an imbalance of osmolytes inside and outside the cell which causes a deviation from the normal turgor. Cells have developed a potent system to deal with this stress in the form of mechanosensitive ion channels. Channel opening releases solutes from the cell and relieves the stress immediately. In bacteria, these channels directly sense the increased membrane tension caused by the enhanced turgor levels upon hypoosmotic shock. The mechanosensitive channel of small conductance, MscS, from Escherichia coli is one of the most extensively studied examples of mechanically stimulated channels. Different conformational states of this channel were obtained in various detergents and membrane mimetics, highlighting an intimate connection between the channel and its lipidic environment. Associated lipids occupy distinct locations and determine the conformational states of MscS. Not all these features are preserved in the larger MscS-like homologues. Recent structures of homologues from bacteria and plants identify common features and differences. This review discusses the current structural and functional models for MscS opening, as well as the influence of certain membrane characteristics on gating.
Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Pressão Osmótica , Membranas/metabolismo , Bactérias/metabolismo , Mecanotransdução CelularRESUMO
The mechanosensitive channel of small conductance (MscS) protects bacteria against hypoosmotic shock. It can sense the tension in the surrounding membrane and releases solutes if the pressure in the cell is getting too high. The membrane contacts MscS at sensor paddles, but lipids also leave the membrane and move along grooves between the paddles to reside as far as 15 Å away from the membrane in hydrophobic pockets. One sensing model suggests that a higher tension pulls lipids from the grooves back to the membrane, which triggers gating. However, it is still unclear to what degree this model accounts for sensing and what contribution the direct interaction of the membrane with the channel has. Here, we show that MscS opens when it is sufficiently delipidated by incubation with the detergent dodecyl-ß-maltoside or the branched detergent lauryl maltose neopentyl glycol. After addition of detergent-solubilized lipids, it closes again. These results support the model that lipid extrusion causes gating: Lipids are slowly removed from the grooves and pockets by the incubation with detergent, which triggers opening. Addition of lipids in micelles allows lipids to migrate back into the pockets, which closes the channel even in the absence of a membrane. Based on the distribution of the aliphatic chains in the open and closed conformation, we propose that during gating, lipids leave the complex on the cytosolic leaflet at the height of highest lateral tension, while on the periplasmic side, lipids flow into gaps, which open between transmembrane helices.
Assuntos
Membrana Celular/fisiologia , Ativação do Canal Iônico/fisiologia , Metabolismo dos Lipídeos , Mecanotransdução Celular/fisiologia , Domínio Catalítico , Lipídeos/química , Modelos Moleculares , Pressão Osmótica , Conformação ProteicaRESUMO
The bacterial mechanosensitive channel of small conductance (MscS) is a well-studied model of how mechanical forces from the membrane can be sensed by an embedded protein. A recent study by Zhang et al. visualizes how MscS behaves under membrane tension, entering a desensitized state when it loses all coordinated lipids.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Modelos MolecularesRESUMO
The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. Escherichia coli has six osmoprotective paralogs with different numbers of transmembrane helices. These helices are important for gating and sensing in MscS but the role of the additional helices in the paralogs is not understood. The medium-sized channel YnaI was extracted and delivered in native nanodiscs in closed-like and open-like conformations using the copolymer diisobutylene/maleic acid (DIBMA) for structural studies. Here we show by electron cryomicroscopy that YnaI has an extended sensor paddle that during gating relocates relative to the pore concomitant with bending of a GGxGG motif in the pore helices. YnaI is the only one of the six paralogs that has this GGxGG motif allowing the sensor paddle to move outward. Access to the pore is through a vestibule on the cytosolic side that is fenestrated by side portals. In YnaI, these portals are obstructed by aromatic side chains but are still fully hydrated and thus support conductance. For comparison with large-sized channels, we determined the structure of YbiO, which showed larger portals and a wider pore with no GGxGG motif. Further in silico comparison of MscS, YnaI, and YbiO highlighted differences in the hydrophobicity and wettability of their pores and vestibule interiors. Thus, MscS-like channels of different sizes have a common core architecture but show different gating mechanisms and fine-tuned conductive properties.
Assuntos
Proteínas de Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Microscopia Crioeletrônica , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Interações Hidrofóbicas e Hidrofílicas , Canais Iônicos/química , Canais Iônicos/ultraestrutura , Metabolismo dos LipídeosRESUMO
Polymeric micelles are typically characterized as core-shell structures. The hydrophobic core is considered as a depot for hydrophobic molecules, and the corona-forming block acts as a stabilizing and solubilizing interface between the core and aqueous milieu. Tremendous efforts have been made to tune the hydrophobic block to increase the drug loading and stability of micelles, whereas the role of hydrophilic blocks is rarely investigated in this context, with poly(ethylene glycol) (PEG) being the gold standard of hydrophilic polymers. To better understand the role of the hydrophilic corona, a small library of structurally similar A-B-A-type amphiphiles based on poly(2-oxazoline)s and poly(2-oxazine)s is investigated by varying the hydrophilic block A utilizing poly(2-methyl-2-oxazoline) (pMeOx; A) or poly(2-ethyl-2-oxazoline) (pEtOx; A*). In terms of hydrophilicity, both polymers closely resemble PEG. The more hydrophobic block B bears either a poly(2-oxazoline) and poly(2-oxazine) backbone with C3 (propyl) and C4 (butyl) side chains. Surprisingly, major differences in loading capacities from A-B-A > A*-B-A > A*-B-A* is observed for the formulation with two poorly water-soluble compounds, curcumin and paclitaxel, highlighting the importance of the hydrophilic corona of polymer micelles used for drug formulation. The formulations are also characterized by various nuclear magnetic resonance spectroscopy methods, dynamic light scattering, cryogenic transmission electron microscopy, and (micro) differential scanning calorimetry. Our findings suggest that the interaction between the hydrophilic block and the guest molecule should be considered an important, but previously largely ignored, factor for the rational design of polymeric micelles.
Assuntos
Portadores de Fármacos/química , Micelas , Oxazóis/química , Polímeros/química , Tensoativos/química , Curcumina/química , Portadores de Fármacos/síntese química , Composição de Medicamentos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Oxazóis/síntese química , Paclitaxel/química , Polímeros/síntese química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Solubilidade , Tensoativos/síntese químicaRESUMO
Amphiphilic block copolymers that undergo (reversible) physical gelation in aqueous media are of great interest in different areas including drug delivery, tissue engineering, regenerative medicine, and biofabrication. We investigated a small library of ABA-type triblock copolymers comprising poly(2-methyl-2-oxazoline) as the hydrophilic shell A and different aromatic poly(2-oxazoline)s and poly(2-oxazine)s cores B in an aqueous solution at different concentrations and temperatures. Interestingly, aqueous solutions of poly(2-methyl-2-oxazoline)-block-poly(2-phenyl-2-oxazine)-block-poly(2-methyl-2-oxazoline) (PMeOx-b-PPheOzi-b-PMeOx) undergo inverse thermogelation below a critical temperature by forming a reversible nanoscale wormlike network. The viscoelastic properties of the resulting gel can be conveniently tailored by the concentration and the polymer composition. Storage moduli of up to 110 kPa could be obtained while the material retains shear-thinning and rapid self-healing properties. We demonstrate three-dimensional (3D) printing of excellently defined and shape-persistent 24-layered scaffolds at different aqueous concentrations to highlight its application potential, e.g., in the research area of biofabrication. A macroporous microstructure, which is stable throughout the printing process, could be confirmed via cryo-scanning electron microscopy (SEM) analysis. The absence of cytotoxicity even at very high concentrations opens a wide range of different applications for this first-in-class material in the field of biomaterials.
RESUMO
Since life has emerged, gradients of osmolytes over the cell membrane cause pressure changes in the cell and require tight regulation to prevent cell rupture. The mechanosensitive channel of small conductance (MscS) releases solutes and water when a hypo-osmotic shock raises the pressure in the cell. It is a member of a large family of MscS-like channels found in bacteria, archaea, fungi and plants and model for mechanosensation. MscS senses the increase of tension in the membrane directly by the force from the lipids, but the molecular mechanism is still elusive. We determined the lipid interactions of MscS by resolving the structure of Escherichia coli MscS embedded in membrane discs to 2.9-Å resolution using cryo-electron microscopy. The membrane is attached only to parts of the sensor paddles of MscS, but phospholipid molecules move through grooves into remote pockets on the cytosolic side. On the periplasmic side, a lipid bound by R88 at the pore entrance is separated from the membrane by TM1 helices. The N-terminus interacts with the periplasmic membrane surface. We demonstrate that the unique membrane domain of MscS promotes deep penetration of lipid molecules and shows multimodal interaction with the membrane to fine-tune tension sensing.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Fenômenos Biofísicos , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Interações Hidrofóbicas e Hidrofílicas , Membranas/metabolismo , Modelos Moleculares , Pressão Osmótica , Fosfolipídeos , Conformação ProteicaRESUMO
Direct electron detectors are an essential asset for the resolution revolution in electron cryo microscopy of biological objects. The direct detectors provide two modes of data acquisition; the counting mode in which single electrons are counted, and the integrating mode in which the signal that arises from the incident electrons is integrated. While counting mode leads to far higher detective quantum efficiency at all spatial frequencies, the integrating mode enables faster data acquisition at higher exposure rates. For optimal throughput at best possible resolution it is important to understand when the better performance in counting mode becomes essential for solving a structure and when the lower detective quantum efficiency in integrating mode can be compensated by increasing the number of particles in the data set. Here, we provide a case study of the Falcon III camera, which has counting mode capability at exposure rates of <0.9 e-/Px² and integrating mode capability at exposure rates above 10 e-/Px². We found that counting mode gives better resolution for medium sized complexes such as the ß-galactosidase (465â¯kDa) (2.2â¯Å, 97% of Nyquist vs. 2.4â¯Å, 89% of Nyquist) with data sets of similar size. However, for larger particles such as Hepatitis B virus capsid like particles (4.8 MDa) we did not find any resolution gain in counting mode.
Assuntos
Microscopia Crioeletrônica/instrumentação , Microscopia Crioeletrônica/métodos , Elétrons , FótonsRESUMO
We present an integrated approach for investigating the topology of proteins through native mass spectrometry (MS) and cross-linking/MS, which we applied to the full-length wild-type p53 tetramer. For the first time, the two techniques were combined in one workflow to obtain not only structural insight in the p53 tetramer, but also information on the cross-linking efficiency and the impact of cross-linker modification on the conformation of an intrinsically disordered protein (IDP). P53 cross-linking was monitored by native MS and as such, our strategy serves as a quality control for different cross-linking reagents. Our approach can be applied to the structural investigation of various protein systems, including IDPs and large protein assemblies, which are challenging to study by the conventional methods used for protein structure characterization.