Controlling Protein Crystal Nucleation by Droplet‐Based Microfluidics

Herein, we demonstrate the potential of droplet‐based microfluidics for controlling protein crystallization and generating single‐protein crystals. We estimated the critical droplet size for obtaining a single crystal within a microdroplet and investigated the crystallization of four model proteins to confirm the effect of protein molecular diffusion on crystallization. A single crystal was obtained in microdroplets smaller than the critical size by using droplet‐based microfluidics. In the case of thaumatin crystallization, a single thaumatin crystal was obtained in a 200 μm droplet even with high supersaturation. In the case of ferritin crystallization, the nucleation profile of ferritin crystals had a wider distribution than the nucleation profiles of lysozyme, thaumatin, and glucose isomerase crystallization. We found that the droplet‐based microfluidic approach was able to control the nucleation of a protein by providing control over the crystallization conditions and the droplet size, and that the diffusion of protein molecules is a significant factor in controlling the nucleation of protein crystals in droplet‐based microfluidics.     

Detergent-associated solution conformations of helical and beta-barrel membrane proteins

Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the beta-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.     

A Robust Microfluidic Device for the Synthesis and Crystal Growth of Organometallic Polymers with Highly Organized Structures

A simple and robust microfluidic device was developed to synthesize organometallic polymers with highly organized structures. The device is compatible with organic solvents. Reactants are loaded into pairs of reservoirs connected by a 15 cm long microchannel prefilled with solvents, thus allowing long‐term counter diffusion for self‐assembly of organometallic polymers. The process can be monitored, and the resulting crystalline polymers are harvested without damage. The device was used to synthesize three insoluble silver acetylides as single crystals of X‐ray diffraction quality. Importantly, for the first time, the single‐crystal structure of silver phenylacetylide was determined. The reported approach may have wide applications, such as crystallization of membrane proteins, synthesis and crystal growth of organic, inorganic, and polymeric coordination compounds, whose single crystals cannot be obtained using traditional methods.     

Modeling detergent organization around aquaporin-0 using small-angle X-ray scattering

Solubilization of integral membrane proteins in aqueous solutions requires the presence of amphiphilic molecules like detergents. The transmembrane region of the proteins is then surrounded by a corona formed by these molecules, ensuring a hydrophilic outer surface. The presence of this corona has strongly hampered structural studies of solubilized membrane proteins by small-angle X-ray scattering (SAXS), a technique frequently used to monitor conformational changes of soluble proteins. Through the online combination of size exclusion chromatography, SAXS, and refractometry, we have determined a precise geometrical model of the n-dodecyl β-d-maltopyranoside corona surrounding aquaporin-0, the most abundant membrane protein of the eye lens. The SAXS data were well-fitted by a detergent corona shaped in an elliptical toroid around the crystal structure of the protein, similar to the elliptical shape recently reported for nanodiscs (Skar-Gislinge et al. J. Am. Chem. Soc. 2010, 132, 13713-13722). The torus thickness determined from the curve-fitting protocol is in excellent agreement with the thickness of a lipid bilayer, while the number of detergent molecules deduced from the volume of the torus compares well with those obtained on the same sample from refractometry and mass analysis based on SAXS forward scattering. For the first time, the partial specific volume of the detergent surrounding a protein was measured. The present protocol is a crucial step toward future conformational studies of membrane proteins in solution.     

基于微流控技术的蛋白质结晶及其筛选方法的研究进展

微流控技术以其高通量、 低消耗和集成化等优点成为蛋白质结晶微型化研究的重要手段. 本文综述了基于微流控技术的蛋白质结晶技术和方法, 主要包括微泵微阀、 液滴(Droplet)、 滑动芯片(SlipChip)以及液滴实验室(DropLab)等技术. 此外, 还针对当前膜蛋白在结构生物学研究中的重要地位, 综述了应用于膜蛋白结晶的微流控技术的研究进展.     

Crowning Proteins: Modulating the Protein Surface Properties using Crown Ethers

Crown ethers are small, cyclic polyethers that have found wide‐spread use in phase‐transfer catalysis and, to a certain degree, in protein chemistry. Crown ethers readily bind metallic and organic cations, including positively charged amino acid side chains. We elucidated the crystal structures of several protein‐crown ether co‐crystals grown in the presence of 18‐crown‐6. We then employed biophysical methods and molecular dynamics simulations to compare these complexes with the corresponding apoproteins and with similar complexes with ring‐shaped low‐molecular‐weight polyethylene glycols. Our studies show that crown ethers can modify protein surface behavior dramatically by stabilizing either intra‐ or intermolecular interactions. Consequently, we propose that crown ethers can be used to modulate a wide variety of protein surface behaviors, such as oligomerization, domain–domain interactions, stabilization in organic solvents, and crystallization.     

基于微流控技术的蛋白质结晶及其筛选方法的研究进展简

微流控技术以其高通量、低消耗和集成化等优点成为蛋白质结晶微型化研究的重要手段. 本文综述了基于微流控技术的蛋白质结晶技术和方法,主要包括微泵微阀、液滴(Droplet)、滑动芯片(SlipChip)以及液滴实验室(DropLab)等技术. 此外,还针对当前膜蛋白在结构生物学研究中的重要地位,综述了应用于膜蛋白结晶的微流控技术的研究进展.     

X-Ray Structure of Bacteriorhodopsin at 2.5 Å Resolution

Microfocussed X-rays produced by an electron synchrotron were essential for the first structure elucidation of protein crystals having extremely small dimensions (5×20–40 μm). A further prerequisite for the successful crystal structure analysis of bacteriorhodopsin was a novel crystallization method using a lipid matrix. These methodological breakthroughs pave the way for further advances in structure determinations of membrane proteins.     

A kinetic model to simulate protein crystal growth in an evaporation-based crystallization platform

The quality, size, and number of protein crystals grown under conditions of continuous solvent extraction are dependent on the rate of solvent extraction and the initial protein and salt concentration. An increase in the rate of solvent extraction leads to a larger number of crystals. The number of crystals decreases, however, when the experiment is started with an initial protein concentration that is closer to the solubility boundary. Here we develop a kinetic model capable of predicting changes in the number and size of protein crystals as a function of time under continuous evaporation. Moreover, this model successfully predicts the initial condition of drops that will result in gel formation. We test this model with experimental crystal growth data of hen egg white lysozyme for which crystal nucleation and growth rate parameters are known from other studies. The predicted and observed rates of crystal growth are in excellent agreement, which suggests that kinetic constants for nucleation and crystal growth for different proteins can be extracted by applying a kinetic model in combination with observations from a few evaporation-based crystallization experiments.     

Membrane protein isolation by in situ solubilization, partitioning and affinity adsorption in aqueous two-phase systems. Purification of the human type 1 11beta-hydroxysteroid dehydrogenase

Recently developed aqueous two-phase systems based on non-ionic detergents and polymers are suitable for the separation of membrane proteins. Moreover, within this relatively membrane protein "friendly" environment, changes in temperature can be controlled and stabilizing agents may be added to ensure integrity of the target protein during isolation. Here, we use aqueous two-phase partitioning for the isolation of membrane bound 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). Different detergents were used to find optimal conditions regarding solubilization and retaining target protein activity. We explored in situ solubilization by adding detergent directly to the aqueous two-phase system, as well as a batch metal affinity capture step of 6xHis tagged 11beta-HSD1 in the two-phase system. The use of detergent/polymer two-phase systems resulted in a specific enzyme activity of 3840 nmol mg(-1) min(-1) of the target membrane protein compared to a conventional purification protocol where a specific enzyme activity of 1440 nmol mg(-1) min(-1) was achieved.     

Noncovalent polymerization of mesogens crystallizes lysozyme: correlation between nonamphiphilic lyotropic liquid crystal phase and protein crystal formation

Crystallization of proteins is important for fundamental studies and biopharmaceutical development but remains largely an empirical science. Here, we report the use of organic salts that can form a class of unusual nonamphiphilic lyotropic liquid crystals to crystallize the protein lysozyme. Certain nonamphiphilic organic molecules with fused aromatic rings and two charges can assemble into stable thread-like noncovalent polymers that may further form liquid crystal phases in water, traditionally termed chromonic liquid crystals. Using five of these mesogenic molecules as additives to induce protein crystallization, we discover that molecules that can form liquid crystal phases in water are highly effective at inducing the crystal formation of lysozyme, even at concentrations significantly lower than that required for forming liquid crystal phases. This result reveals an example of inducing protein crystallization by the molecular assembly of the additives, and is consistent with a new mechanism by which the strong hydration of an assembly process provides a gradual means to compete for the water molecules to enable solvated proteins to form crystals.     

Using microfluidics to observe the effect of mixing on nucleation of protein crystals

This paper analyzes the effect of mixing on nucleation of protein crystals. The mixing of protein and precipitant was controlled by changing the flow rate in a plug-based microfluidic system. The nucleation rate inversely depended on the flow rate, and flow rate could be used to control nucleation. For example, at higher supersaturations, precipitation happened at low flow rates while large crystals grew at high flow rates. Mixing at low flow velocities in a winding channel induces nucleation more effectively than mixing in straight channels. A qualitative scaling argument that relies on a number of assumptions is presented to understand the experimental results. In addition to helping fundamental understanding, this result may be used to control nucleation, using rapid chaotic mixing to eliminate formation of precipitates at high supersaturation and using slow chaotic mixing to induce nucleation at lower supersaturation.     

Tuning in-meso-crystallized lysozyme polymorphism by lyotropic liquid crystal symmetry

Lipid-based lyotropic liquid crystals (LLCs) show great potential for applications in fields as diverse as food technology, cosmetics, pharmaceutics, or structural biology. Recently, these systems have provided a viable alternative to the difficult process of membrane protein crystallization, owing to their similarities with cell membranes. Nonetheless, the process of in-meso crystallization of proteins still remains poorly understood. In this study, we demonstrate that in-meso crystal morphologies of lysozyme (LSZ), a model hydrophilic protein, can be controlled by both the composition and symmetry of the mesophase, inferring a possible general influence of the LLC space group on the protein crystal polymorphism. Lysozyme was crystallized in-meso from three common LLC phases (lamellar, inverse hexagonal, and inverse bicontinuous cubic) composed of monolinolein and water. Different mixing ratios of mesophase to crystallization buffer were used in order to tune crystallization both in the bulk mesophase and in excess water conditions. Two distinct mechanisms of crystallization were shown to take place depending on available water in the mesophases. In the bulk mesophases, protein nuclei form and grow within structural defects of the mesophase and partially dehydrate the system inducing order-to-order transitions of the liquid crystalline phase toward stable symmetries in conditions of lower hydration. The formed protein crystals eventually macrophase separate from the mesophase allowing the system to reach its final symmetry. On the other hand, when excess water is available, protein molecules diffuse from the water channels into the excess water, where the crystallization process can take place freely, and with little to no effect on the structure and symmetry of the lyotropic liquid crystals.     

Growth rates of protein crystals

Protein crystallization is important for structural biology. The rate at which a protein crystallizes is often the bottleneck in determining the protein's structure. Here, we give a physical model for the growth rates of protein crystals. Most materials crystallize faster under stronger growth conditions; however, protein crystallization slows down under the strongest conditions. Proteins require a crystallization slot of 'just right' conditions. Our model provides an explanation. Unlike simpler materials, proteins are orientationally asymmetrical. Under strong conditions, protein molecules attempt to crystallize too quickly, in wrong orientations, blocking surface sites for more productive crystal growth. The model explains the observation that increasing the net charge on a protein increases the crystal growth rate. The model predictions are in good agreement with experiments on the growth rates of tetragonal lysozyme crystals as a function of pH, salt concentration, temperature, and protein concentration.     

SOLUTION CRYSTALLIZATION OF METALLOCENE SHORT CHAIN BRANCHED POLYETHYLENE:MORPHOLOGY AND MECHANISM*

Solution crystallization of metallocene short chain branched polyethylene (SCBPE) was carried out and very nicesingle crystals were obtained. Compared with single crystals grown from linear polyethylene, SCBPE single crystals are dirtydue to intermolecular heterogeneity The crystal morphology changes with crystallization temperatures. Lozenge, truncatedlozenge, hexagonal, rounded and elongated crystal morphologies have been found at much lower crystallization temperaturethan in linear polyethylene. The electron diffraction shows there is a possibility that the single crystals may have hexagonalpacking in a crystallization temperature range. The lateral habits of single crystal are discussed based on roughening theories.     

基于渗透脱水的自动化蛋白质结晶高通量筛选芯片

构建了一种基于渗透脱水模式的自动进样微流控结晶芯片. 该芯片通过真空预脱气将包含蛋白质和结晶剂的液滴自动分配至结晶微腔阵列中, 然后利用集成的一排包含不同浓度盐溶液的透析管道, 通过渗透脱水方式经一层聚二甲基硅氧烷(PDMS)膜实现液滴的逐渐浓缩, 使之趋于过饱和状态, 进而形成结晶. 此芯片可一次筛选较宽范围的过饱和状态, 实现蛋白质结晶的快速优化. 利用模式蛋白溶菌酶的结晶实验验证了该芯片的性能.     

Screening of C60 crystallization using a microfluidic system

We have carried out screening of C60 crystallization using a simple liquid/liquid interfacial precipitation method in a microfluidic device. By controlling the time, temperature, and concentration, various metastable phases of C60 crystals were found, including tubes, spheres, open-ended hollow columns, stars, branches, and trees. The obtained C60 crystal shapes are similar to those of snow crystals. These findings suggest an urgent need to screen C60 crystallization for the development of fullerene C60 drugs.     

A microfluidic device for kinetic optimization of protein crystallization and in situ structure determination

The unprecedented economies of scale and unique mass transport properties of microfluidic devices made them viable nano-volume protein crystallization screening platforms. However, realizing the full potential of microfluidic crystallization requires complementary technologies for crystal optimization and harvesting. In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through "kinetic optimization" of crystallization reactions and in situ structure determination. Kinetic optimization through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in-chip.     

Rapid, highly efficient extraction and purification of membrane proteins using a microfluidic continuous-flow based aqueous two-phase system

Membrane proteins play essential roles in regulating various fundamental cellular functions. To investigate membrane proteins, extraction and purification are usually prerequisite steps. Here, we demonstrated a microfluidic aqueous PEG/detergent two-phase system for the purification of membrane proteins from crude cell extract, which replaced the conventional discontinuous agitation method with continuous extraction in laminar flows, resulting in significantly increased extraction speed and efficiency. To evaluate this system, different separation and detection methods were used to identify the purified proteins, such as capillary electrophoresis, SDS-PAGE and nano-HPLC-MS/MS. Swiss-Prot database with Mascot search engine was used to search for membrane proteins from random selected bands of SDS-PAGE. Results indicated that efficient purification of membrane proteins can be achieved within 5-7s and approximately 90% of the purified proteins were membrane proteins (the highest extraction efficiency reported up to date), including membrane-associated proteins and integral membrane proteins with multiple transmembrane domains. Compared to conventional approaches, this new method had advantages of greater specific surface area, minimal emulsification, reduced sample consumption and analysis time. We expect the developed method to be potentially useful in membrane protein purifications, facilitating the investigation of membrane proteomics.