Self-assembled lipid and membrane protein polyhedral nanoparticles

We demonstrate that membrane proteins and phospholipids can self-assemble into polyhedral arrangements suitable for structural analysis. Using the Escherichia coli mechanosensitive channel of small conductance (MscS) as a model protein, we prepared membrane protein polyhedral nanoparticles (MPPNs) with uniform radii of ∼20 nm. Electron cryotomographic analysis established that these MPPNs contain 24 MscS heptamers related by octahedral symmetry. Subsequent single-particle electron cryomicroscopy yielded a reconstruction at ∼1-nm resolution, revealing a conformation closely resembling the nonconducting state. The generality of this approach has been addressed by the successful preparation of MPPNs for two unrelated proteins, the mechanosensitive channel of large conductance and the connexon Cx26, using a recently devised microfluidics-based free interface diffusion system. MPPNs provide not only a starting point for the structural analysis of membrane proteins in a phospholipid environment, but their closed surfaces should facilitate studies in the presence of physiological transmembrane gradients, in addition to potential applications as drug delivery carriers or as templates for inorganic nanoparticle formation.The functions of many membrane proteins are intimately coupled to the generation, utilization, and/or sensing of transmembrane gradients (1). Despite advances in the structure determination of membrane proteins (2), the high-resolution structural analysis of membrane proteins in a biological membrane is uncommon and in the presence of a functionally relevant gradient remains an as-yet unrealized experimental challenge. This stems from the fact that the primary 2D- and 3D ordered specimens used in structural studies of membrane proteins by X-ray crystallography and electron microscopy lack closed membrane surfaces, thus making it impossible to establish physiologically relevant transmembrane gradients.As an alternative, we have been developing methodologies for the self-assembly of lipids and membrane proteins into closed polyhedral structures that can potentially support transmembrane gradients for structural and functional studies. The possibility of generating polyhedral arrangements of membrane proteins in proteoliposomes was motivated by the existence of polyhedral capsids of membrane-enveloped viruses (3, 4), the ability of surfactant mixtures to self-assemble into polyhedral structures (5, 6), and the formation of proteoliposomes from native membranes containing bacteriorhodopsin (7, 8) and light-harvesting complex II (LHCII) (9). Significantly, the high-resolution structure of LHCII was determined from crystals of icosahedral proteoliposomes composed of protein subunits in chloroplast lipids (10). Whereas detergent solubilized membrane proteins and lipid mixtures can self-assemble to form 2D-ordered crystalline sheets or helical tubes favorable for structure determination by electron microscopy (11–14), simple polyhedral ordered assemblies have only been described to form from select native membranes (7–9). To expand the repertoire of membrane protein structural methods, we have prepared membrane protein polyhedral nanoparticles (MPPNs) of the bacterial mechanosensitive channel of small conductance (MscS) (15, 16) from detergent solubilized protein and phospholipids, and demonstrated that they are amenable to structural analysis using electron microscopy.Conditions for generating MPPNs were anticipated to resemble those for other types of 2D-ordered bilayer arrangements of membrane proteins, particularly 2D crystals, in that membrane protein is mixed with a particular phospholipid at a defined ratio, followed by dialysis to remove the solubilizing detergent (17). The main distinction is that because MPPNs are polyhedral, conditions are sought that will stabilize highly curved surfaces of polyhedra rather than the planar (flat) specimens desired for 2D crystals. We used the Escherichia coli MscS as a model system. MscS is an intrinsically stretch-activated channel identified by Booth and coworkers (15) that confers resistance to osmotic downshock in E. coli. MscS forms a heptameric channel with 21 transmembrane helices (3 from each subunit) and a large cytoplasmic domain with overall dimensions of ∼8 × ∼12 nm parallel and perpendicular to the membrane plane; structures have been reported in both nonconducting (16, 18) and open-state conformations (19). Different phospholipids were added to purified the E. coli MscS solubilized in the detergent Fos-Choline 14 and the system was allowed to reach equilibrium by dialysis at different temperatures. To gain insight into the biophysical parameters that govern MPPN formation, we investigated the role of lipid head group, alkyl chain length, pH, and protein construct. Table S1 shows the observed influence of these various factors on our ability to form uniform MPPNs (as opposed to disordered aggregates or polydisperse proteoliposomes). The optimal conditions for MPPN formation used 1,2-dimyristoyl-sn-glycero-3-phosphocholine [added to ∼1:0.1 (wt/wt) protein:phospholipid] at pH 7 with the His-tagged MscS that is anticipated to be positively charged under these conditions. The biophysical properties of the protein are important as the best results were achieved using a His-tagged construct and the presence of a FLAG tag at the C terminus of MscS interfered with MPPN formation, even though the tag is ∼10 nm from the membrane-spanning region of MscS.To monitor MPPN formation, dynamic light scattering (DLS) was used. Under optimal conditions, we observed (Fig. 1A) the complete transition of solubilized MscS particles with a narrow distribution centered around a mean radii of 4.5 nm to MPPNs with a narrow distribution centered around a mean radii of 20 nm. We further characterized these particles using negative-stain electron microscopy. Fig. 1B is a field view negative-stain electron micrograph of a solution of detergent-solubilized MscS and lipid before initiation of the self-assembly process. Fig. 1C is a field view negative-stain electron micrograph of the same sample after the self-assembly process. We observed the incorporation of MscS into highly uniform polyhedra with mean radii of ∼20 nm (90%) and ∼17 nm (10%) by negative-stain electron microscopy. To gain more insight into the biophysical properties of these particles, we performed protein and phosphorus analysis on multiple samples to determine the lipid:protein ratio (Fig. S1). The observed lipid:protein ratio of the MscS MPPNs was 11 ± 1 (mole lipid:mole protein subunit) and consistent with a single layer of lipids forming a bilayer surrounding each protein. This ratio is comparable to the observed lipid to protein ratio found in 2D crystals of membrane proteins such as bacteriorhodopsin (lipid:protein ratio of 10; refs. 20 and 21) and aquaporin (lipid:protein ratio of 9; ref. 22).Open in a separate windowFig. 1.Preparation of MscS MPPNs. (A) DLS analysis of particles before dialysis and after completion of dialysis when MPPNs are formed. The observed radius of MscS alone was 4.5 nm and the particle radius at the end of dialysis was observed to be 20 nm. In both cases 99% of the scattering mass was observed in the distributions centered at 4.5 nm and 20 nm, respectively. (B) Negative-stain electron microscopy analysis of MscS before dialysis. Individual MscS proteins can be observed as small doughnut-shaped particles. (C) Negative-stain electron microscopy analysis of MPPNs following dialysis of the sample in B. MPPNs can be clearly observed and appear as uniform assemblies of individual MscS molecules. (Scale bars, 100 nm.)To further elucidate the structural nature of these particles and to unambiguously determine the symmetry, we performed electron cryotomography with image reconstruction using IMOD (23) combined with Particle Estimation for Electron Tomography (PEET) program (ref. 24 and SI Materials and Methods). In principle, electron tomography provides a complete 3D map of the particles and would allow us to unambiguously determine the MPPN symmetry. However, the alignment process was highly biased by the missing wedge phenomenon (24) due to poor signal:noise and resulted in an incomplete map (Fig. 2A). To overcome this alignment bias, we assigned random initial orientation values to all particles and constrained possible angular shifts to less than 30° to achieve a more uniform distribution of orientations (Fig. S2). This strategy resulted in a much improved density map (Fig. 2B) that revealed individual molecules with a size and shape that are in good agreement to the known molecular structure of MscS (Fig. 2C and Fig. S3). Building on the analysis of Haselwandter and Phillips (25), a systematic analysis was conducted (Table S2) of the symmetry relationships between MscSs in MPPNs that identified the arrangement corresponding to the snub cuboctahedron (dextro), an Archimedean solid. The snub cuboctahedron has cubic (octahedral) symmetry which, as recognized by Crick and Watson (26), provides an efficient way to pack identical particles in a closed, convex shell. In this particular arrangement, 24 MscS molecules are related by the 432-point group symmetry axes that pass through the faces, but not the vertices, of the snub cuboctahedron. Because the MscS molecules are positioned on the vertices of this chiral polyhedron, they occupy general positions that permit the ordered packing of the heptamers of a biomacromolecule (or indeed any type of particle). This is an important observation as it means that the individual MscS molecules with sevenfold symmetry are capable of packing into a symmetric assembly that is amenable to averaging. Whereas 24 objects can be arranged with identical environments in a snub cuboctahedron, certain integer multiples of this number can also be accommodated using the principles of quasiequivalence (27, 28) to form larger closed shells.Open in a separate windowFig. 2.Cryotomography of MscS MPPNs. (A) The PEET isosurface derived from 162 individual particles selected from eight single-tilt tomograms. The strong bias due to the missing wedge is observed along the lower part of the surface, but individual MscS heptamers are still discernible in the image. (B) The corresponding PEET isosurface, following introduction of randomized starting Euler angles to minimize missing-wedge bias. (C) The use of randomized starting Euler angles results in a much-improved map with apparent octahedral (432) symmetry that could be fit with 24 molecules of the MscS crystal structure. Isosurface renderings of the volume averages were generated using Chimera (31).Using the symmetry derived by electron cryotomography, we proceeded to collect high-resolution single-particle electron cryomicroscopy data. Samples prepared identically for cryotomography were imaged under low-dose conditions and a total of 4,564 particles were processed using the Electron Micrograph Analysis 2 (EMAN2) software package (SI Materials and Methods) (29). The final map had a resolution of 9 Å by Fourier shell correlation (Fig. S4) and allowed us to model the inner and outer helices of the transmembrane pore (Fig. 3). The arrangement of the helices more closely resembles the nonconducting conformation (16, 18) than the open-state structure (19), although some differences in the positioning of the outer helices relative to the nonconducting structure are indicated in sections 2 and 3 of Fig. 3. These results demonstrate that membrane proteins are capable of assembling into MPPNs that are amenable to high-resolution structure analysis by single-particle electron cryomicroscopy. Higher resolution data will be required, however, to detail the precise conformational differences between MscS in the phospholipid environment of MPPNs compared with those in the detergent-solubilized state used in the X-ray crystal structure analyses.Open in a separate windowFig. 3.Single-particle image analysis reconstructed from 4,564 particles processed with EMAN2 and subsequently the density surrounding a single MscS heptamer was extracted and sevenfold averaged as described in SI Materials and Methods. (Left) A cross-section through the electron density revealing the translocation pathway and cytoplasmic vestibule, and showing the overall fit of the closed structure of MscS (red ribbons) fit to the map (cyan). (Right) Stereoviews of cross-sections in the density map normal to the sevenfold axis at sections 1, 2, and 3. The closed-structure coordinates (red ribbons) of MscS were fit to the map using rigid body refinement in Chimera (31) showing the position of the transmembrane helices.In these promising initial studies we used traditional dialysis methods to screen conditions for MPPN formation. These methods are time consuming and require substantial quantities of a sample. To more efficiently screen conditions for MPPN formation with a variety of membrane proteins, we designed and fabricated a free interface diffusion microfluidic device (30) (Fig. 4A and Fig. S5) This device greatly simplifies the screening process and minimizes the amount of sample required for determining suitable conditions for MPPN formation. Using this device, we were able to produce MPPNs from MscS but more importantly from several other proteins that had previously failed to produce MPPNs using traditional dialysis. Fig. 4 B and C shows the results of using this device for the mechanosensitive channel of large conductance (MscL) and the connexon Cx26, respectively, where polyhedra were only observed in the presence of the target protein. Intriguingly, several different particles sizes could be observed for both MscL and Cx26 and we hypothesize that the variable-sized polyhedra may correspond to different packing arrangements similar to triangulation numbers observed in viral polyhedral assemblies. This microfluidic device will provide rapid screening of conditions for the formation of MPPNs and it is hoped will expedite membrane protein structural analysis in native lipid environments.Open in a separate windowFig. 4.Preparation of MPPNs using a microfluidics-based free interface diffusion system. (A) Schematic illustration of the device used for lipid–protein nanoparticle formation. From left to right, molecules in the center flow diffuse into the outer flow by the concentration gradient, with small molecules (larger diffusion coefficient) moving more quickly than larger molecules. Specifically, monomer detergents are removed through interfacial diffusion, whereas larger membrane proteins remain in the center flow, forming nanoparticles. Both the ratio of input:buffer and the flow rate influence particle formation. (B). Negative-stain electron microscopy images of MPPNs of MscL and (C) Cx26 formed using the microfluidic device from A. (Scale bar, 100 nm.) Insets show 2.5× magnification of a select region of interest.The self-assembly of membrane proteins into polyhedral nanoparticles demonstrates a potentially powerful method for studying the structure and function of membrane proteins in a lipid environment. MPPNs represent a novel form of lipid–protein assemblies which lie between single particles and large crystalline sheets or tubes. We have demonstrated that conditions favorable for MPPN formation can be identified and have elucidated the structure, symmetry, and potential application to membrane protein structure analysis. In addition we have designed and fabricated microfluidic devices for high-throughput screening of conditions for MPPN formation. MPPNs may allow a variety of perturbations to be achieved such as pH, voltage, osmotic, concentration gradients, etc. that cannot be achieved with other membrane protein assemblies and will potentially allow us to activate various types of gated channels and receptors so that active conformational states can be structurally investigated. The potential of such materials for targeted drug delivery with precisely controlled release mechanisms offers an intriguing avenue for future biomedical applications.     

Stigma predicts residential treatment length for substance use disorder

Background: Stigma has been suggested as a possible contributor to the high rates of treatment attrition in substance-dependent individuals, but no published empirical studies have examined this association. Objectives: The present paper assessed the relationship between baseline stigma variables and length of treatment stay in a sample of patients in a residential addictions treatment unit. Methods: The relationship between baseline stigma variables (self-stigma, enacted stigma, and shame) and length of stay for participants (n?=?103) in a residential addictions treatment unit was examined. Results: Higher self-stigma predicted longer stay in residential addictions treatment, even after controlling for age, marital status, race, overall mental health, social support, enacted stigma, and internalized shame. However, other stigma variables (i.e. internalized shame, stigma-related rejection) did not reliably predict length of treatment stay. Conclusion: These results are consistent with other findings suggesting that people with higher self-stigma may have a lowered sense of self-efficacy and heightened fear of being stigmatized and therefore retreat into more protected settings such as residential treatment, potentially resulting in higher treatment costs. Specialized clinical interventions may be necessary to help participants cope with reduced self-efficacy and fear of being stigmatized.     

Psychometric properties of the Creole Haitian version of the Resilience Scale amongst child and adolescent survivors of the 2010 earthquake

Resilience is defined as the capacity of human beings to deal with and adapt to adversity, suffering, tragedy or other traumatic event. This study aims to investigate psychometric properties and the underlying structure of the Creole version of the RS among children and adolescents survivors to the 2010 Haitian earthquake. A total of 872 children and adolescents exposed to the earthquake with an average age of 14.91 (SD = 1.94) completed the Creole version of RS, the Impact Event Scale-Revised, the Children Depression Inventory and the Social Support Questionnaire-6. The current validity of RS and the internal consistency were investigated; sex, age, religion and others socio-demographic variables differences were also analysed. Cronbach’s alpha coefficient for the RS was .77; the split-half coefficient was .72. The goodness-of-fit for the 5-factor model presents the best adjusted indices. The total resilience score was correlated positively with social support (r = .42, p < .01). Mean score of the RS was 131.46 (SD = 21.01). No significant differences were observed about sex, age and residential municipality. The results showed that the Haitian Creole version of RS is a valid and reliable measure in assessing resilience for the children and adolescent survivors to the 2010’s earthquake.     

Variable temporoinsular cortex neuroanatomy in primates suggests a bottleneck effect in eastern gorillas

We describe an atypical neuroanatomical feature present in several primate species that involves a fusion between the temporal lobe (often including Heschl's gyrus in great apes) and the posterior dorsal insula, such that a portion of insular cortex forms an isolated pocket medial to the Sylvian fissure. We assessed the frequency of this fusion in 56 primate species (including apes, Old World monkeys, New World monkeys, and strepsirrhines) by using either magnetic resonance images or histological sections. A fusion between temporal cortex and posterior insula was present in 22 species (seven apes, two Old World monkeys, four New World monkeys, and nine strepsirrhines). The temporoinsular fusion was observed in most eastern gorilla (Gorilla beringei beringei and G. b. graueri) specimens (62% and 100% of cases, respectively) but was seen less frequently in other great apes and was never found in humans. We further explored the histology of this fusion in eastern gorillas by examining the cyto‐ and myeloarchitecture within this region and observed that the degree to which deep cortical layers and white matter are incorporated into the fusion varies among individuals within a species. We suggest that fusion between temporal and insular cortex is an example of a relatively rare neuroanatomical feature that has become more common in eastern gorillas, possibly as the result of a population bottleneck effect. Characterizing the phylogenetic distribution of this morphology highlights a derived feature of these great apes. J. Comp. Neurol. 522:844–860, 2014. © 2013 Wiley Periodicals, Inc.     

Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase

Systemic lupus erythematosus (SLE), the prototypic systemic autoimmune disease, is a debilitating multisystem autoimmune disorder characterized by chronic inflammation and extensive immune dysregulation in multiple organ systems, resulting in significant morbidity and mortality. Here, we present a multidisciplinary approach resulting in the identification of neutrophil cytosolic factor 2 (NCF2) as an important risk factor for SLE and the detailed characterization of its causal variant. We show that NCF2 is strongly associated with increased SLE risk in two independent populations: childhood-onset SLE and adult-onset SLE. The association between NCF2 and SLE can be attributed to a single nonsynonymous coding mutation in exon 12, the effect of which is the substitution of histidine-389 with glutamine (H389Q) in the PB1 domain of the NCF2 protein, with glutamine being the risk allele. Computational modeling suggests that the NCF2 H389Q mutation reduces the binding efficiency of NCF2 with the guanine nucleotide exchange factor Vav1. The model predicts that NCF2/H389 residue interacts with Vav1 residues E509, N510, E556, and G559 in the ZF domain of Vav1. Furthermore, replacing H389 with Q results in 1.5 kcal/mol weaker binding. To examine the effect of the NCF2 H389Q mutation on NADPH oxidase function, site-specific mutations at the 389 position in NCF2 were tested. Results show that an H389Q mutation causes a twofold decrease in reactive oxygen species production induced by the activation of the Vav-dependent Fcγ receptor-elicited NADPH oxidase activity. Our study completes the chain of evidence from genetic association to specific molecular function.     

Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein

Cilia and flagella are highly conserved motile and sensory organelles in eukaryotes, and defects in ciliary assembly and motility cause many ciliopathies. The two-headed I1 inner arm dynein is a critical regulator of ciliary and flagellar beating. To understand I1 architecture and function better, we analyzed the 3D structure and composition of the I1 dynein in Chlamydomonas axonemes by cryoelectron tomography and subtomogram averaging. Our data revealed several connections from the I1 dynein to neighboring structures that are likely to be important for assembly and/or regulation, including a tether linking one I1 motor domain to the doublet microtubule and doublet-specific differences potentially contributing to the asymmetrical distribution of dynein activity required for ciliary beating. We also imaged three I1 mutants and analyzed their polypeptide composition using 2D gel-based proteomics. Structural and biochemical comparisons revealed the likely location of the regulatory IC138 phosphoprotein and its associated subcomplex. Overall, our studies demonstrate that I1 dynein is connected to multiple structures within the axoneme, and therefore ideally positioned to integrate signals that regulate ciliary motility.