Molecular fluorescence blinking provides a simple and attractive way to achieve super-resolution localization via conventional fluorescence microscopy. However, success in super-resolution imaging relies heavily on their blinking characteristics. We here report easily prepared and photostable nanoparticles, carbon dots (CDs), with desirable fluorescence blinking for high-density super-resolution imaging. The CDs exhibit a low duty cycle (∼0.003) and high photon output (∼8000) per switching event, as well as show much higher resistance to photobleaching than Alexa 647 or Cy5 typically used in single molecule localization microscopy. The stable blinking of CDs allows to perform high-density localization imaging at a resolution of 25 nm by sequentially recording the particle positions. The CD-based super-resolution imaging is further demonstrated by rendering CD-stained tubular peptide self-assemblies, CD-packed clusters with well-defined patterns, and CD-stained microtubules in a cell. Furthermore, this method has been validated as a valuable tool to detect the clustering and distribution of protein receptors in the plasma membrane that are not discerned with normal fluorescence imaging.

Nucleoli are important subnuclear structures inside cells. We report novel fluorescent gold nanoclusters (K–AuNCs) that are able to stain the nucleoli selectively and make it possible to explore the nucleolar morphology with fluorescence imaging technique. This novel probe is prepared through an easy synthesis method by employing a tripeptide (Lys–Cys–Lys) as the surface ligand. The properties, including deep-red fluorescence emission (680 nm), large Stocks shift, broad excitation band, low cytotoxicity, and good photostability, endow this probe with potential for bioanalytical applications. Because of their small size and their positively charged surface, K–AuNCs are able to accumulate efficiently at the nucleolar regions and provide precise morphological information. K–AuNCs are also used to monitor the nucleolar dynamics along the reverse-transformation process of malignant cells, induced by the agonist of protein A, 8-chloro-cyclic adenosine monophosphate. This gives a novel approach for investigating the working mechanism of antitumor drugs.Keywords: bioimaging; fluorescence; gold nanoclusters; nucleolus; reverse transformation;

Taking inspiration from biology’s effectiveness in functionalizing protein-based nanocages for chemical processes, we describe here a rational design of an artificial metalloenzyme for oxidations with the bacterial chaperonin GroEL, a nanocage for protein folding in nature, by supramolecular anchoring of catalytically active hemin in its hydrophobic central cavity. The promiscuity of the chaperonin cavity is an essential element of this design, which can mimic the hydrophobic binding pocket in natural metalloenzymes to accept cofactor and substrate without requiring specific ligand–protein interactions. The success of this approach is manifested in the efficient loading of multiple monomeric hemin cofactors to the GroEL cavity by detergent dialysis and good catalytic oxidation properties of the resulting biohybrid in tandem with those of the clean oxidant of H2O2. Investigation of the mechanism of hemin–GroEL-catalyzed oxidation of two-model substrates reveals that the kinetic behavior of the complex follows a ping-pong mechanism in both cases. Through comparison with horseradish peroxidase, the oxidative activity and stability of hemin–GroEL were observed to be similar to those found in natural peroxidases. Adenosine 5′-triphosphate (ATP)-regulated partial dissociation of the biohybrid, as assessed by the reduction of its catalytic activity with the addition of the nucleotide, raises the prospect that ATP may be used to recycle the chaperonin scaffold. Moreover, hemin–GroEL can be applied to the chromogenic detection of H2O2, which (or peroxide in general) is commonly contained in industrial wastes. Considering the rich chemistry of free metalloporphyrins and the ease of production of GroEL and its supramolecular complex with hemin, this work should seed the creation of many new artificial metalloenzymes with diverse reactivities.Keywords: artificial metalloenzyme; catalytic material; chaperonin; hemin; oxidation; protein nanocage;

The role of dimerization and oligomerization of G-protein-coupled receptors in their signal transduction is highly controversial. Delineating this issue can greatly facilitate rational drug design. With single-molecule imaging, we show that chemokine receptor CXCR4 exists mainly as a monomer in normal mammalian living cells and forms dimers and higher-order oligomers at a high expression level, such as in cancer cells. Chemotaxis tests demonstrate that the signal transduction activity of CXCR4 does not depend only on its expression level, indicating a close relation with the oligomeric status of CXCR4. Moreover, binding ligands can effectively upregulate or downregulate the oligomeric level of CXCR4, which suggests that binding ligands may realize their pivotal roles by regulating the oligomeric status of CXCR4 rather than by simply inducing conformational changes.

The aggregation of amyloid beta (Aβ) peptides plays a crucial role in the pathology and etiology of Alzheimer's disease. Experimental evidence shows that copper ion is an aggregation-prone species with the ability to coordinately bind to Aβ and further induce the formation of neurotoxic Aβ oligomers. However, the detailed structures of Cu(II)–Aβ complexes have not been illustrated, and the kinetics and dynamics of the Cu(II) binding are not well understood. Two Cu(II)–Aβ complexes have been proposed to exist under physiological conditions, and another two might exist at higher pH values. By using ab initio simulations for the spontaneous resonance Raman and time domain stimulated resonance Raman spectroscopy signals, we obtained the characteristic Raman vibronic features of each complex. These signals contain rich structural information with high temporal resolution, enabling the characterization of transient states during the fast Cu–Aβ binding and interconversion processes.

The nucleolus is an important subnuclear structure and there are very few dyes available in the market for nucleolar imaging. Subcellular organelles delivery presents a common stumbling block for many nanomaterial-based applications, including intracellular structure fluorescence staining. We now introduce a novel luminescent graphene quantum dot (nGQD), which is able to selectively light up the nucleoli of living cells, to address these challenges. Investigations on subcellular localization of different GQDs have demonstrated that the positively charged surface and the ultra-small size are the key parameters for nucleoli-rich distribution of nanomaterials, which is of importance for transferring this strategy to other nanoparticles. The novel nGQD has great potential to be applied as a nucleolar stain or an efficient gene/drug carrier.

Fluorescence imaging requires bioselective, sensitive, nontoxic molecular probes to detect the precise location of lesions for fundamental research and clinical applications. Typical inorganic semiconductor nanomaterials with large sizes (>10 nm) can offer high-quality fluorescence imaging due to their fascinating optical properties but are limited to low selectivity as well as slow clearance pathway. We here report an N- and O-rich carbogenic small molecular complex (SMC, MW < 1000 Da) that exhibits high quantum yield (up to 80%), nucleic acid-binding enhanced excitation-dependent fluorescence (EDF), and a near-infrared (NIR) emission peaked at 850 nm with an ultralarge Stokes shift (∼500 nm). SMCs show strong rRNA affinity, and the resulting EDF enhancement allows multicolor visualization of nucleoli in cells for clear statistics. Furthermore, SMCs can be efficiently accumulated in tumor in vivo after injection into tumor-bearing mice. The NIR emission affords high signal/noise ratio imaging for delineating the true extent of tumor. Importantly, about 80% of injected SMCs can be rapidly excreted from the body in 24 h. No appreciable toxicological responses were observed up to 30 days by hematological, biochemical, and pathological examinations. SMCs have great potential as a promising nucleolus- and tumor-specific agent for medical diagnoses and biomedical research.Keywords: excitation-dependent fluorescence; RNA selectivity; small molecule; target-specific imaging; ultralarge Stokes shift

We present novel active targeting luminescent gold nanoclusters (AuNCs), which are prepared through a one-pot procedure by using a pentapeptide (CRGDS) for stabilization and tumor recognition. CRGDS–AuNCs exhibit a high tumor-specific retention with an exceptionally high tumor-to-liver uptake ratio of 9.3. Their small hydrodynamic diameter and zwitterionic surface facilitate urinary excretion, which reaches 82% within 24 h after injection.

Increasing evidence indicates that carbon nanoparticles (CNPs), which mainly originate from incomplete combustion of fossil fuels, have an adverse impact on the respiratory system. Recent in vivo experiments have shown that the pulmonary toxicity of CNPs is attributed to their aggregation in pulmonary surfactant monolayers (PSMs) while the underlying mechanism of aggregation remains unclear. Here, by performing coarse grained molecular dynamics simulations, we demonstrate for the first time that the aggregation of carbon nanospheres (CNSs) in PSMs is in fact size-dependent and mediated by lipid extractions. Upon CNS deposition, neighbouring lipid molecules are extracted from PSMs to cover CNSs from the top side. The extracted lipids induce clustering of CNSs to maximize the CNS–lipid interaction, by forming inverse micelles to wrap the aggregated CNSs cooperatively. The formed CNS clusters perturb the molecule structure of the PSM and thus affect its biofunction on respiration. Our simulations show that during the expiration process, CNSs form clusters that perturb the mechanical properties of the PSM in a manner depending on the CNS size. With deep inspiration, a high concentration of large CNSs may induce PSM rupture and thus have a potential impact on its biophysical properties.

How soft tubular aggregates interact with biomembranes is crucial for understanding the formation of membrane tubes connecting two eukaryotic cells, which are initially created from one cell and then connect with the other. On the other hand, recent experiments have shown that tubular polymersomes display different cellular internalization kinetics in their biomedical applications compared with spherical ones with an underlying mechanism that is not fully understood. Inspired by above observations, in this work we investigate how tubular aggregates interact with biomembranes with the aid of computer simulation techniques. We identify three different pathways for membrane interaction with parallel tubes: membrane wrapping, tube-membrane fusion and tube pearling. For the first pathway, soft tubes can be wrapped from the top side by membranes through membrane monolayer protrusion, which cooperatively leads to a heterogeneous wrapping dynamics along with tube deformation. The second pathway found is that soft tubes fuse with the membrane under certain conditions. Both wrapping and fusion have distinct influence on the third pathway, tube pearling. While a weak membrane adhesion promotes tube pearling, the strong adhesion that leads to higher extent of membrane wrapping conversely restrains tube pearling. Under highly positive membrane tension, partial tube-membrane fusion provides another way to mediate tube pearling. The findings shed light on the formation of a bridge membrane tube and the rational design of tube-based therapeutic agents with improved efficiency for targeted cellular delivery.

A common mechanism for intracellular transport is the controlled shape transformation, also known as pearling, of membrane tubes. Exploring how tube pearling takes place is thus of quite importance to not only understand the bio-functions of tubes, but also promote their potential biomedical applications. While the pearling mechanism of one single tube is well understood, both the pathway and the mechanism of pearling of multiple tubes still remain unclear. Herein, by means of computer simulations we show that the tube pearling can be mediated by the inter-tube adhesion. By increasing the inter-tube adhesion strength, each tube undergoes a discontinuous transition from no pearling to thorough pearling. The discontinuous pearling transition is ascribed to the competitive variation between tube surface tension and the extent of inter-tube adhesion. Besides, the final pearling instability is also affected by tube diameter and inter-tube orientation. Thinner tubes undergo inter-tube lipid diffusion before completion of pearling. The early lipid diffusion reduces the extent of inter-tube adhesion and thus restrains the subsequent pearling. Therefore, only partial or no pearling can take place for two thinner tubes. For two perpendicular tubes, the pearling is also observed, but with different pathways and higher efficiency. The finite size effect is discussed by comparing the pearling of tubes with different lengths. It is expected that this work will not only provide new insights into the mechanism of membrane tube pearling, but also shed light on the potential applications in biomaterials science and nanomedicine.

After the synthesis of transmembrane peptides/proteins (TMPs), their insertion into a lipid bilayer is a fundamental biophysical process. Moreover, correct orientations of TMPs in membranes determine the normal functions they play in relevant cellular activities. In this study, we have established a method to determine the orientation of TMPs in membranes. This method is based on the use of TAMRA, a fluorescent molecule with high extinction coefficient and fluorescence quantum yield, to act as a fluorescent probe and tryptophan as a quencher. Fluorescence quenching indicates that the model peptide displays membrane orientation with the N terminus outside and the C terminus inside dominantly. To elucidate the underlying mechanism, we performed molecular dynamics simulations. Our simulations suggest that both membrane insertion and the orientation of TMPs are determined by complex competition and cooperation between hydrophobic and electrostatic interactions. After initial membrane anchorage via electrostatic interactions of the charged residues with the lipid headgroups, further insertion is hindered by unfavorable interactions between the polar residues and lipid tails, which result in an energy barrier. Nevertheless, such a finite energy barrier is reduced by hydrophobic interactions between the non-polar residues and lipid tails. Moreover, a transient terminal flipping was captured to facilitate the membrane insertion. Once the inserted terminus reaches the opposite lipid headgroups, the hydrophobic interactions cooperate with the electrostatic interactions to complete the membrane insertion process.

The receptor-mediated endocytosis of nanoparticles (NPs) is known to be size and shape dependent but regulated by membrane properties, like tension, rigidity, and especially membrane proteins. Compared with transmembrane receptors, which directly bind ligands coated on NPs to provide the driving force for passive endocytosis, the hidden role of inner anchored membrane proteins (IAMPs), however, has been grossly neglected. Here, by applying the N-varied dissipative particle dynamics (DPD) techniques, we present the first simulation study on the interplay between wrapping of NPs and clustering of IAMPs. Our results suggest that the wrapping dynamics of NPs can be regulated by clustering of IAMPs, but in a competitive way. In the early stage, the dispersed IAMPs rigidify the membrane and thus restrain NP wrapping by increasing the membrane bending energy. However, once the clustering completes, the rigidifying effect is reduced. Interestingly, the clustering of longer IAMPs can sense NP wrapping. They are found to locate preferentially at the boundary region of NP wrapping. More importantly, the adjacent IAMP clustering produces a late membrane monolayer protrusion, which finally wraps the NP from the top side. Our findings regarding the competitive effects of IAMP clustering on NP wrapping facilitate the molecular understanding of endocytosis and establish fundamental principles for design of NPs for widespread biomedical applications.

Using nanotechnology, therapeutics can be combined with diagnostics for cancer treatment. To do this, a targeting ligand, an imaging contrast agent and an anti-tumour therapeutic agent were the minimum requirements for active targeting nanoassemblies. Here we have developed a novel active targeting theranostic agent, made up of just two components, aptamer AS1411 and graphene quantum dots (GQDs). Each component in our agent plays multiple roles. Confocal microscopy using a 488 nm laser shows that this agent has an excellent capability to label tumour cells selectively. On the therapeutic side, this agent induced a synergistic growth inhibition effect towards cancer cells when irradiated with a near infrared laser of 808 nm. The ultra-small size, good biocompatibility, intrinsic stable fluorescence, and near-infrared response character make GQDs a remarkable constituent to build theranostic agents.

Recently, a unique dynamic magnetic field was developed to induce the rotational movement of superparamagnetic iron oxide nanoparticles. This technique has been applied to remotely control both cellular internalization and apoptosis. Therefore, a thorough understanding of how a lipid membrane responds to the introduction of rotating NPs is quite important to promote the applications of this technique in a variety of biomedical area. Here, we performed Dissipative Particle Dynamics (DPD) simulations to systematically investigate the interaction mechanism between lipid membranes and rotating NPs. Two kinds of membrane responses are observed. One is the promoted cell uptake and the other is the mechanical membrane rupture. The promoting effect of NP rotation on the cell uptake is ascribed to the enhanced membrane monolayer protrusion, which can wrap the NP from the top side. Meanwhile, the rotating NP exerts a shearing force on the membrane. Accordingly, the membrane undergoes a local distortion around the NP. If the shearing force exceeds a critical value, the local membrane distortion develops into a mechanical rupture. A number of factors, like NP size, NP shape, ligand density and rotation speed, are critical in both of the above membrane responses.

Understanding how nanoparticles interact with the pulmonary surfactant monolayer (PSM) is of great importance for safe applications in biomedicine and for evaluation of both health and environment impacts. Here, by performing molecular dynamics simulations, we propose a possible origin of the pulmonary nanotoxicity of graphene-based nanoparticles that comes from a rigidifying effect of graphene nanosheets (GNs) on PSM. This, in reality, indicates that once captured by the PSM, inhaled GNs are hard to be removed from the PSM partially because the expiration or PSM compression is locally restrained, possibly leading to GN accumulation on the PSM. The local rigidifying effect, which is enhanced as multiple GNs approach each other, is found to be dependent on the GN hydrophobicity. In the expiration or PSM compression process, the hydrophilic GN keeps adhering to the monolayer–air interface, while the hydrophobic GN tends to be hosted in the hydrophobic interior and internalize into the PSM via self-rotation. Besides the spontaneous internalization via PSM compression, our pulling simulations indicate that both pulmonary internalization and externalization of GNs can be accomplished by direct translocation across the PSM. The effect of GN hydrophobicity on the direct PSM translocation is well supported by the free energy analysis. This work will help our understanding of pulmonary nanotoxicity of GNs and provide useful guidelines for molecular design of GN-based pulmonary drug delivery materials.

Gigaporous polystyrene (PS) microspheres were hydrophilized by in situ polymerization to give a stable cross-linked poly(vinyl alcohol) (PVA) hydrogel coating, which can shield proteins from the hydrophobic PS surface underneath. The amination of microspheres (PS-NH2) was first carried out through acetylization, oximation and reduction, and then 4,4′-azobis (4-cyanovaleric acid) (ACV), a polymerization initiator, was covalently immobilized on PS-NH2 through amide bond formation, and the cross-linked poly(vinyl acetate) (PVAc) was prepared by radical polymerization at the surfaces of ACV-immobilized PS microspheres (PS-ACV). Finally, the cross-linked PVA hydrogel coated gigaporous PS microspheres (PS-PVA) was easily achieved through alcoholysis of PVAc. Results suggested that the PS microspheres were effectively coated with cross-linked PVA hydrogel, where the gigaporrous structure remained under optimal conditions. After hydrophilic modification (PS-PVA), the protein-resistant ability of microspheres was greatly improved. The hydroxyl-rich PS-PVA surface can be easily derivatized by classical chemical methods. Performance advantages of the PS-PVA column in flow experiment include good permeability, low backpressure, and mechanical stability. These results indicated that PS-PVA should be promising in rapid protein chromatography.Keywords: coating; cross-linked poly(vinyl alcohol) hydrogel; gigaporous; polystyrene particles; protein adsorption

The shape transformation of membrane tubes, also known as pearling, is thought to play an important role in a variety of cellular activities, like intracellular transport. Despite considerable experiments have investigating this phenomenon, the detailed molecular mechanism as well as how environmental factors affect the tube pearling instability is still ambiguous. In this work, we use computer simulation techniques to obtain a molecular-level insight into the tube pearling process. We find that the tube morphology is strongly determined by the water pressure inside membrane tubes. For example, the tube shrinkage and subsequent bending is observed when we decrease the inner water pressure. Contrarily, as we increase the inner water pressure, the tube pearling tends to occur in order to reduce the surface energy. Besides, our simulations show that the membrane tube pearling is regulated by the adsorption of nanoparticles (NPs) in two competing ways. One is that the NP adsorption can exert an additional membrane tension and thus promote the pearling and subsequent division of membrane tubes. On the other hand, the NP adsorption can locally rigidify the membrane and thus contrarily restrain the tube pearling. Therefore, the NP size, NP concentration and NP-membrane adhesion strength will collectively regulate the tube pearling process.

Understanding how nanoparticles (NPs) interact with the lipid membrane is of importance for their potential applications in biomedicine and cytotoxic effects. In this paper, with the aid of computer simulation techniques, we report that NPs can be wrapped by lipid membranes in a pathway different from the conventional endocytic pathway. Our simulation results show that under the conditions of strong NP–membrane adhesion and low membrane tension, NPs can be wrapped by membranes with a pathway regulated by membrane monolayer protrusion. We also find that in the monolayer protrusion mediated wrapping pathway NPs are first trapped in the membrane and the subsequent NP internalization can be achieved by several means, including decreasing the membrane tension, breaking the membrane symmetry between upper and lower leaflets, and exerting an external force on the NPs. The findings from our simulations are well supported by the free energy analysis.

•Modified graphene quantum dots are applied as a multifunctional drug carrier.•Multiple tasks are carried out simultaneously.•Cellular uptake of the nanoassembly and drug release are monitored in real time.•The nanoassembly targets tumor cells differentially and efficiently.•The nanoassembly exhibits significantly reduced cytotoxicity to non-target cells.This study demonstrates that ligand-modified graphene quantum dots (GQDs) facilitate the simultaneous operation of multiple tasks without the need for external dyes. These tasks include selective cell labeling, targeted drug delivery, and real-time monitoring of cellular uptake. Folic acid (FA)-conjugated GQDs are synthesized and utilized to load the antitumor drug doxorubicin (DOX). The fabricated nanoassembly can unambiguously discriminate cancer cells from normal cells and efficiently deliver the drug to targeted cells. The inherent stable fluorescence of GQDs enables real-time monitoring of the cellular uptake of the DOX–GQD–FA nanoassembly and the consequent release of drugs. The nanoassembly is specifically internalized rapidly by HeLa cells via receptor-mediated endocytosis, whereas DOX release and accumulation are prolonged. In vitro toxicity data suggest that the DOX–GQD–FA nanoassembly can target HeLa cells differentially and efficiently while exhibiting significantly reduced cytotoxicity to non-target cells.

Surfactants play a significant role in solubilization of photosystem I (PSI) in vitro. Triton X-100 (TX), n-Dodecyl-β-d-maltoside (DDM), and sodium dodecyl sulfate (SDS) were employed to solubilize PSI particles in MES buffer to compare the effect of surfactant and its dosage on the apparent oxygen consumption rate of PSI. Through a combined assessment of sucrose density gradient centrifugation, Native PAGE and 77 K fluorescence with the apparent oxygen consumption, the nature of the enhancement of the apparent oxygen consumption activity of PSI by surfactants has been analyzed. Aggregated PSI particles can be dispersed by surfactant molecules into micelles, and the apparent oxygen consumption rate is higher for surfactant-solubilized PSI than for integral PSI particles. For DDM, PSI particles are solubilized mostly as the integral trimeric form. For TX, PSI particles are solubilized as incomplete trimeric and some monomeric forms. For the much harsher surfactant, SDS, PSI particles are completely solubilized as monomeric and its subunit forms. The enhancement of the oxygen consumption rate cannot be explained only by the effects of surfactant on the equilibrium between monomeric and trimeric forms of solubililized PSI. Care must be taken when the electron transfer activity of PSI is evaluated by methods based on oxygen consumption because the apparent oxygen consumption rate is influenced by uncoupled chlorophyll (Chl) from PSI, i.e., the larger the amount of uncoupled Chl, the higher the rate of apparent oxygen consumption. 77 K fluorescence spectra can be used to ensure that there is no uncoupled Chl present in the system. In order to eliminate the effect of trace uncoupled Chl, an efficient physical quencher of 1O2, such as 1 mM NaN3, may be added into the mixture.

Surfactants play important roles in the preparation, structural, and functional research of membrane proteins, and solubilizing and isolating membrane protein, while keeping their structural integrity and activity intact is complicated. The commercial n-Dodecyl-β-D-maltoside (DDM) and Triton X-100 (TX) were used as solubilizers to extract and purify trimeric photosystem I (PSI) complex, an important photosynthetic membrane protein complex attracting broad interests. With an optimized procedure, TX can be used as an effective surfactant to isolate and purify PSI, as a replace of the much more expensive DDM. A mechanism was proposed to interpret the solubilization process at surfactant concentrations lower than the critical solubilization concentration. PSI-TX and PSI-DDM had identical polypeptide bands, pigment compositions, oxygen consumption, and photocurrent activities. This provides an alternative procedure and paves a way for economical and large-scale trimeric PSI preparation.

Genes of the key enzymes for phycocyanobilin (PCB) biosynthesis were cloned into E. coli and combinationally expressed to produce phycocyanobilin, with autologous heme as substrate. Culture conditions were optimized to achieve ~3 mg PCB/l. A protocol for the purification of recombinant phycocyanobilin was established using solvent extraction combined with chromatography, which resulted in a final yield of ~0.3 mg PCB/l with a purity >95 %. Recombinant phycocyanobilin could scavenge hydroxyl radicals with an EC50 of 0.1 μM.

•Synergies of the bacterial chaperonin GroEL-GroES and cell-free expression for the production of functionally folded CXCR4 is studied.•GroEL-GroES greatly increases the rate and yield of CXCR4 functional folding.•The structural stability and ligand binding affinity of CXCR4 can be improved with supplied GroEL-GroES.•The cooperation between GroEL and GroES is required to promote efficient folding.•New insights into membrane protein production and folding, as well as the role of molecular chaperones.G protein-coupled receptors (GPCRs) are important therapeutic targets for a broad spectrum of diseases and disorders. Obtaining milligram quantities of functional receptors through the development of robust production methods are highly demanded to probe GPCR structure and functions. In this study, we analyzed synergies of the bacterial chaperonin GroEL-GroES and cell-free expression for the production of functionally folded C-X-C chemokine GPCR type 4 (CXCR4). The yield of soluble CXCR4 in the presence of detergent Brij-35 reached ∼1.1 mg/ml. The chaperonin complex added was found to significantly enhance the productive folding of newly synthesized CXCR4, by increasing both the rate (∼30-fold) and the yield (∼1.3-fold) of folding over its spontaneous behavior. Meanwhile, the structural stability of CXCR4 was also improved with supplied GroEL-GroES, as was the soluble expression of biologically active CXCR4 with a ∼1.4-fold increase. The improved stability together with the higher ligand binding affinity suggests more efficient folding. The essential chaperonin GroEL was shown to be partially effective on its own, but for maximum efficiency both GroEL and its co-chaperonin GroES were necessary. The method reported here should prove generally useful for cell-free production of large amounts of natively folded GPCRs, and even other classes of membrane proteins.

•The barnacle adopts a disc saw-like base perforated with radial microchannels.•The base is constructed of intergrown micro-calcites with unique features.•Both proteins and polysaccharides are involved in the microstructural development.•Barnacle base has been shown to adjust over different scales to enhance adhesion.How the calcareous bases of acorn barnacles are interfacially structured and conserved for underwater attachment function remains unclear, especially given the existence of proteinaceous adhesives in between the bases and substrata. We demonstrate with the barnacle Balanus albicostatus that the base adopts a unique, disc saw-like geometry, densely perforated with radial microchannels that are intelligently built by the animal to enhance adhesion. Meanwhile, the base is shown to be constructed of intergrown calcitic microcrystals with almost no preferred orientation, some varied extent of morphologic change and high level of atomic disorder compared to its geological equivalent. Further, both protein and polysaccharide chemistries are found to be involved in the microstructural development of the base by virtue of their acidic functional groups or confined space, with the latter (behaving as a hydrogel) possibly being dominant in amount. The key microstructural features along with the placement of organic components play crucial roles in the mechanical response of the base to external loads. Our results explain how the barnacle base has been evolutionarily adjusted over different length scales to enhance adhesion, which would lay the foundation of research into antifouling strategy as well as the synthesis of organic-inorganic hybrid interfacial materials.

We present novel active targeting luminescent gold nanoclusters (AuNCs), which are prepared through a one-pot procedure by using a pentapeptide (CRGDS) for stabilization and tumor recognition. CRGDS–AuNCs exhibit a high tumor-specific retention with an exceptionally high tumor-to-liver uptake ratio of 9.3. Their small hydrodynamic diameter and zwitterionic surface facilitate urinary excretion, which reaches 82% within 24 h after injection.

Using nanotechnology, therapeutics can be combined with diagnostics for cancer treatment. To do this, a targeting ligand, an imaging contrast agent and an anti-tumour therapeutic agent were the minimum requirements for active targeting nanoassemblies. Here we have developed a novel active targeting theranostic agent, made up of just two components, aptamer AS1411 and graphene quantum dots (GQDs). Each component in our agent plays multiple roles. Confocal microscopy using a 488 nm laser shows that this agent has an excellent capability to label tumour cells selectively. On the therapeutic side, this agent induced a synergistic growth inhibition effect towards cancer cells when irradiated with a near infrared laser of 808 nm. The ultra-small size, good biocompatibility, intrinsic stable fluorescence, and near-infrared response character make GQDs a remarkable constituent to build theranostic agents.

After the synthesis of transmembrane peptides/proteins (TMPs), their insertion into a lipid bilayer is a fundamental biophysical process. Moreover, correct orientations of TMPs in membranes determine the normal functions they play in relevant cellular activities. In this study, we have established a method to determine the orientation of TMPs in membranes. This method is based on the use of TAMRA, a fluorescent molecule with high extinction coefficient and fluorescence quantum yield, to act as a fluorescent probe and tryptophan as a quencher. Fluorescence quenching indicates that the model peptide displays membrane orientation with the N terminus outside and the C terminus inside dominantly. To elucidate the underlying mechanism, we performed molecular dynamics simulations. Our simulations suggest that both membrane insertion and the orientation of TMPs are determined by complex competition and cooperation between hydrophobic and electrostatic interactions. After initial membrane anchorage via electrostatic interactions of the charged residues with the lipid headgroups, further insertion is hindered by unfavorable interactions between the polar residues and lipid tails, which result in an energy barrier. Nevertheless, such a finite energy barrier is reduced by hydrophobic interactions between the non-polar residues and lipid tails. Moreover, a transient terminal flipping was captured to facilitate the membrane insertion. Once the inserted terminus reaches the opposite lipid headgroups, the hydrophobic interactions cooperate with the electrostatic interactions to complete the membrane insertion process.

The shape transformation of membrane tubes, also known as pearling, is thought to play an important role in a variety of cellular activities, like intracellular transport. Despite considerable experiments have investigating this phenomenon, the detailed molecular mechanism as well as how environmental factors affect the tube pearling instability is still ambiguous. In this work, we use computer simulation techniques to obtain a molecular-level insight into the tube pearling process. We find that the tube morphology is strongly determined by the water pressure inside membrane tubes. For example, the tube shrinkage and subsequent bending is observed when we decrease the inner water pressure. Contrarily, as we increase the inner water pressure, the tube pearling tends to occur in order to reduce the surface energy. Besides, our simulations show that the membrane tube pearling is regulated by the adsorption of nanoparticles (NPs) in two competing ways. One is that the NP adsorption can exert an additional membrane tension and thus promote the pearling and subsequent division of membrane tubes. On the other hand, the NP adsorption can locally rigidify the membrane and thus contrarily restrain the tube pearling. Therefore, the NP size, NP concentration and NP-membrane adhesion strength will collectively regulate the tube pearling process.

Increasing evidence indicates that carbon nanoparticles (CNPs), which mainly originate from incomplete combustion of fossil fuels, have an adverse impact on the respiratory system. Recent in vivo experiments have shown that the pulmonary toxicity of CNPs is attributed to their aggregation in pulmonary surfactant monolayers (PSMs) while the underlying mechanism of aggregation remains unclear. Here, by performing coarse grained molecular dynamics simulations, we demonstrate for the first time that the aggregation of carbon nanospheres (CNSs) in PSMs is in fact size-dependent and mediated by lipid extractions. Upon CNS deposition, neighbouring lipid molecules are extracted from PSMs to cover CNSs from the top side. The extracted lipids induce clustering of CNSs to maximize the CNS–lipid interaction, by forming inverse micelles to wrap the aggregated CNSs cooperatively. The formed CNS clusters perturb the molecule structure of the PSM and thus affect its biofunction on respiration. Our simulations show that during the expiration process, CNSs form clusters that perturb the mechanical properties of the PSM in a manner depending on the CNS size. With deep inspiration, a high concentration of large CNSs may induce PSM rupture and thus have a potential impact on its biophysical properties.

How soft tubular aggregates interact with biomembranes is crucial for understanding the formation of membrane tubes connecting two eukaryotic cells, which are initially created from one cell and then connect with the other. On the other hand, recent experiments have shown that tubular polymersomes display different cellular internalization kinetics in their biomedical applications compared with spherical ones with an underlying mechanism that is not fully understood. Inspired by above observations, in this work we investigate how tubular aggregates interact with biomembranes with the aid of computer simulation techniques. We identify three different pathways for membrane interaction with parallel tubes: membrane wrapping, tube-membrane fusion and tube pearling. For the first pathway, soft tubes can be wrapped from the top side by membranes through membrane monolayer protrusion, which cooperatively leads to a heterogeneous wrapping dynamics along with tube deformation. The second pathway found is that soft tubes fuse with the membrane under certain conditions. Both wrapping and fusion have distinct influence on the third pathway, tube pearling. While a weak membrane adhesion promotes tube pearling, the strong adhesion that leads to higher extent of membrane wrapping conversely restrains tube pearling. Under highly positive membrane tension, partial tube-membrane fusion provides another way to mediate tube pearling. The findings shed light on the formation of a bridge membrane tube and the rational design of tube-based therapeutic agents with improved efficiency for targeted cellular delivery.

A common mechanism for intracellular transport is the controlled shape transformation, also known as pearling, of membrane tubes. Exploring how tube pearling takes place is thus of quite importance to not only understand the bio-functions of tubes, but also promote their potential biomedical applications. While the pearling mechanism of one single tube is well understood, both the pathway and the mechanism of pearling of multiple tubes still remain unclear. Herein, by means of computer simulations we show that the tube pearling can be mediated by the inter-tube adhesion. By increasing the inter-tube adhesion strength, each tube undergoes a discontinuous transition from no pearling to thorough pearling. The discontinuous pearling transition is ascribed to the competitive variation between tube surface tension and the extent of inter-tube adhesion. Besides, the final pearling instability is also affected by tube diameter and inter-tube orientation. Thinner tubes undergo inter-tube lipid diffusion before completion of pearling. The early lipid diffusion reduces the extent of inter-tube adhesion and thus restrains the subsequent pearling. Therefore, only partial or no pearling can take place for two thinner tubes. For two perpendicular tubes, the pearling is also observed, but with different pathways and higher efficiency. The finite size effect is discussed by comparing the pearling of tubes with different lengths. It is expected that this work will not only provide new insights into the mechanism of membrane tube pearling, but also shed light on the potential applications in biomaterials science and nanomedicine.

The nucleolus is an important subnuclear structure and there are very few dyes available in the market for nucleolar imaging. Subcellular organelles delivery presents a common stumbling block for many nanomaterial-based applications, including intracellular structure fluorescence staining. We now introduce a novel luminescent graphene quantum dot (nGQD), which is able to selectively light up the nucleoli of living cells, to address these challenges. Investigations on subcellular localization of different GQDs have demonstrated that the positively charged surface and the ultra-small size are the key parameters for nucleoli-rich distribution of nanomaterials, which is of importance for transferring this strategy to other nanoparticles. The novel nGQD has great potential to be applied as a nucleolar stain or an efficient gene/drug carrier.