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.     

基于细胞水平药物筛选的微流控芯片系统研究进展

进入21世纪以来,药物筛选技术正朝着快速、高效的细胞水平筛选方面发展,微流控芯片技术作为现代生命科学领域的重要研究工具,以其分析微型化、高通量化、可集成化和良好的生物相容性等特点,在细胞水平药物筛选方面引起了广泛关注,并取得了一系列成果。国家“十二五”规划中启动科技重大专项“基于微流控芯片的新药研究开发关键技术”,使得微流控芯片在药物筛选领域的研究达到了一个新的高度。本文主要综述了近年来基于细胞水平药物筛选的微流控芯片系统的研究进展,并对其应用前景进行了展望。     

Disclosing respiratory co-infections: a broad-range panel assay for avian respiratory pathogens on a nanofluidic PCR platform

Respiratory syndromes (RS) are among the most significant pathological conditions in edible birds and are caused by complex coactions of pathogens and environmental factors. In poultry, low pathogenic avian influenza A viruses, metapneumoviruses, infectious bronchitis virus, infectious laryngotracheitis virus, Mycoplasma spp. Escherichia coli and/or Ornithobacterium rhinotracheale in turkeys are considered as key co-infectious agents of RS. Aspergillus sp., Pasteurella multocida, Avibacterium paragallinarum or Chlamydia psittaci may also be involved in respiratory outbreaks. An innovative quantitative PCR method, based on a nanofluidic technology, has the ability to screen up to 96 samples with 96 pathogen-specific PCR primers, at the same time, in one run of real-time quantitative PCR. This platform was used for the screening of avian respiratory pathogens: 15 respiratory agents, including viruses, bacteria and fungi potentially associated with respiratory infections of poultry, were targeted. Primers were designed and validated for SYBR green real-time quantitative PCR and subsequently validated on the Biomark high throughput PCR nanofluidic platform (Fluidigm©, San Francisco, CA, USA). As a clinical assessment, tracheal swabs were sampled from turkeys showing RS and submitted to this panel assay. Beside systematic detection of E. coli, avian metapneumovirus, Mycoplasma gallisepticum and Mycoplasma synoviae were frequently detected, with distinctive co-infection patterns between French and Moroccan flocks. This proof-of-concept study illustrates the potential of such panel assays for unveiling respiratory co-infection profiles in poultry.     

Dispersion of Dynamic Biochemical Signals in Steady Flow in a Shallow Y-type Microfluidic Channel

This paper presents an analysis of dispersion of dynamic biochemical signals in steady flow in a shallow Y-type microfluidic channel. A method is presented to control the flow widths of two steady flows in the Y-type microchannel from two inlets. The transfer function for the Y-type microchannel is given by solving the governing e- quation for the Taylor-Aris dispersion in the microchannel. The amplitude-frequency and phase-frequency relations are provided which show that a shallow Y-type mi- crochannel acts as a low-pass filter. The transports of different dynamic biochemical sig- nals are investigated. In comparison with a fully mixing microfluidic channel, the magni- tudes of the dynamic signals at the outlets in a Y-type microchannel are much smaller than those in a fully mixing microchannel, which demonstrates that the amplitude atten- uation in a Y-type microchannel is larger than that of a fully mixing microchannel due to the transverse molecular diffusion. In order to control the desired signal in a microchannel, the solution of the inverse problem for the channel is also presented.     

体外仿生三维血管生成微流控芯片的构建

目的在微流控芯片上构建模拟人体血管的三维血管管道,实现管道间的物质交换,并形成有自主出芽功能的血管,为血管生成相关的疾病机制研究、药物筛选等提供良好的平台工具。方法利用微流控技术,采用被动进样方式,使人体脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)在微流控芯片上自主贴壁生长形成三维血管管腔,构建体外仿生三维单管道血管和双管道血管芯片。在此基础上,开展对血管响应刺激因子出芽的功能的验证实验。结果体外仿生的双管道芯片的构建成功率达80%以上。在血管管道,内皮细胞无需借助外力支撑,在芯片上自主形成直径300μm±50μm,细胞分布均匀的、类似体内血管的三维血管管腔;物质通过模拟体内的扩散过程达到血管管道,使血管管腔响应物质刺激而出现功能性的血管出芽。结论体外仿生三维血管微流控芯片在体外实现了体内血管的结构与功能,模拟了体内物质扩散对血管生成的影响,可以用于生理过程中血管生成的体外动态观察。     

微流控蛋白印迹杂交与传统Western blot对目的蛋白的检测效果比较

目的借助微流控蛋白印迹实验实现对目的蛋白省时、省力和省试剂的定性以及相对定量分析。方法选取O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)蛋白为目的蛋白,选用高表达MGMT蛋白的MCF7细胞为细胞模型,用传统蛋白印迹杂交和纸芯片蛋白印迹杂交实验法对经过药物MGMT阻断剂(lomeguatrib)处理后MCF7细胞的MGMT蛋白表达量做定性以及相对定量分析。结果纸芯片蛋白印迹杂交可以成功进行MGMT的定性、相对定量分析,而且上样检测量可低至10~25 pg。这相比于传统Western blot的上样检测蛋白检测限1~5 ng,提高了3个数量级。整个操作流程只需要1 h,所用试剂量少,实验成本更低。而且,纸芯片的多通道构造可以实现如同反向蛋白杂交分析(RPPA)一样的,对组织蛋白表达量进行高通量的系统检测。结论微流控蛋白印迹杂交相较于传统Western blot可以检测到更微量级的蛋白,节省试剂和样品,省时和省力,并可进行高通量的定性以及相对定量分析。     

Batch‐mode microfluidic radiosynthesis of N‐succinimidyl‐4‐[18F]fluorobenzoate for protein labelling

The batch microfluidic technology is a promising system for sequential chemical steps combining the advantages of micro‐scale reactions, while addressing some shortcomings of flow‐through systems. We report herein the convenient three‐step, one‐pot synthesis and purification of [¹⁸F]SFB. [¹⁸F]SFB is a radiolabelling agent that can be used to label sensitive biomolecules, which are not accessible by direct nucleophilic ¹⁸F‐fluorination. Five sequential steps were performed with a batch microfluidic device to obtain the short‐lived positron‐emitter‐labelled molecule. Aqueous [¹⁸F]fluoride was concentrated and further eluted to a microreactor for evaporation. Nucleophilic ¹⁸F‐fluorination of the precursor was carried out at high temperature, prior to hydrolysis and subsequent activation of the 4‐[¹⁸F]fluorobenzoyl group. Purification on miniaturized solid‐phase finally afforded [¹⁸F]SFB in 25 min and 55±6% yield (not decay‐corrected) and >98% radiochemical purity. In this study, microfluidic prepared [¹⁸F]SFB could be further successfully used for labelling the epidermal growth factor protein. These results illustrate how microfluidic batch devices are advantageous for producing radiotracers for molecular imaging, e.g. Positron emission tomography. The technology offers many benefits such as the possibility to use much smaller quantities of starting material, reduced reaction time combined with improved efficiency, and easier purification. Copyright © 2010 John Wiley & Sons, Ltd.     

Current and emerging trends in point-of-care urinalysis tests

ABSTRACT

Introduction: The development of point-of-care testing (POCT) has made clinical diagnostics available, affordable, rapid, and easy to use since the 1990s.The significance of this platform rests on its potential to empower patients to monitor their own health status more frequently, in the convenience of their home, so that diseases can be diagnosed at the earliest possible time-point. Recent advances have expanded traditional formats such as qualitative or semi-quantitative dipsticks and lateral flow immunoassays to newer platforms such as microfluidics and paper-based assays where signals can be measured quantitatively using handheld devices.

Areas covered: This review discusses: (1) working principles and operating mechanisms of both existing and emerging POCT platforms, (2) urine analytes measured using POCT in comparison to the laboratory or clinical ‘gold standard,’ and (3) limitations of existing POCT and expectations of emerging POCT in urinalysis.

Expert opinion: Currently, a variety of biological samples such as urine, saliva, serum, plasma, and other fluids can be applied to POCT for quick diagnosis, especially in resource-limited settings. Emerging platforms will increasingly empower individuals to monitor their health status through frequent urine analysis even from their homes. The impact of these emerging technologies on healthcare is likely to be transformative.     

微流控芯片精子分选法对精子常规参数及DNA完整性的影响

目的:研究微流控芯片精子优选技术对精子常规参数及DNA完整性的影响。方法:自行设计制作微流控芯片,利用芯片处理技术和上游法对40例精液标本进行精子优选,通过计算机辅助精液分析系统及染色质扩散试验从精子常规参数及DNA完整性两个方面评价精液体外处理对精子的影响。结果:精液经微流控芯片法和上游法处理后,精子活力、精子正常形态率以及精子尾部肿胀率均有显著提高(P<0.01),精子的DNA损伤率明显降低(P<0.01)。微流控芯片法与上游法相比,优选后前者精子DNA损伤率明显低于后者[(8.4±5.8)%vs(16.4±9.2)%,P<0.01],而其他参数差异无显著性。结论:微流控芯片技术在精子优选中能获得精子DNA损伤程度小的高质量精子。     

应用电化学测菌法检测变形链球菌的初步探讨

目的:探讨应用电化学测菌法检测变形链球菌的可行性。方法:利用本课题组前期建立的电化学测菌法检测变形链球菌,记录检测参数,并将电化学测菌法的检测结果与细菌培养结果进行比较。结果:①微流芯片样本通道内层流形成时间为(3.17±0.16)min,从进样到获得稳定电阻抗数据的时间为(10.20±0.16)min。②当变形链球菌浓度为104～109cells/mL时,细菌浓度C的对数与系统阻抗值Z呈良好的线性关系(R2=0.98)。③与细菌培养的检测结果相比,电化学测菌法获得的细菌数多,差异有统计学意义(P<0.05)。④变形链球菌浓度为105cells/mL时,电化学测菌法检测值与细菌培养法检测值之间的线性函数关系式为:y=0.94x+0.32E4(R2=0.95),两种方法的检测值高度正相关(P=0.001<0.01)。结论:电化学测菌法能为变形链球菌的快速检测提供新的思路。