A Plug-Based Microfluidic System for Dispensing Lipidic Cubic Phase (LCP) Material Validated by Crystallizing Membrane Proteins in Lipidic Mesophases

This article presents a plug-based microfluidic system to dispense nanoliter-volume plugs of lipidic cubic phase (LCP) material and subsequently merge the LCP plugs with aqueous plugs. This system was validated by crystallizing membrane proteins in lipidic mesophases, including LCP. This system allows for accurate dispensing of LCP material in nanoliter volumes, prevents inadvertent phase transitions that may occur due to dehydration by enclosing LCP in plugs, and is compatible with the traditional method of forming LCP material using a membrane protein sample, as shown by the successful crystallization of bacteriorhodopsin from Halobacterium salinarum. Conditions for the formation of LCP plugs were characterized and presented in a phase diagram. This system was also implemented using two different methods of introducing the membrane protein: (1) the traditional method of generating the LCP material using a membrane protein sample and (2) post LCP-formation incorporation (PLI), which involves making LCP material without protein, adding the membrane protein sample externally to the LCP material, and allowing the protein to diffuse into the LCP material or into other lipidic mesophases that may result from phase transitions. Crystals of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis were obtained using PLI. The plug-based, LCP-assisted microfluidic system, combined with the PLI method for introducing membrane protein into LCP, should be useful for minimizing consumption of samples and broadening the screening of parameter space in membrane protein crystallization

Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins

We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time

Microfluidic platforms for membrane protein crystallization and in situ crystallography

Membrane proteins, biological macromolecules that reside in cellular membranes, play critical roles in many biological process, including signaling, transport, and intercellular communication. The malfunction of membrane proteins has been linked to the initiation or progression of many diseases (e.g. autism, diabetes), so the study of their precise structure is of critical interest in the field of drug discovery and structure-based drug design. Structure-based drug development relies on the knowledge of atomic resolution 3D protein structures, the relationship between protein function and structure, and how these proteins interact with potential drug molecules. X ray crystallography, presently the most common and robust method for solving structures, relies on the growth of high quality protein crystals. The structural pipeline reaches a bottleneck during X-ray crystallography because conditions for protein crystallization cannot be determined a priori – extensive screening methods (i.e. trial and error) across a multi-parametric chemical space must be conducted to discover appropriate crystallization conditions using limited amounts of precious membrane protein sample. Analysis of eukaryotic genomes that 30% of all proteins are membrane proteins, however <2% of all known structures are membrane proteins. Given their significant role in disease and the slow pace of structure elucidation, new methods are needed to accelerate structure discovery for membrane proteins. The membrane protein crystallization toolbox contains many powerful, yet difficult-to-use tools. For example, nucleation and growth are typically coupled during crystallization experiments, which limits the degree of control over the quality and size of crystals grown in solution. Seeding techniques, where crystals grow from existing nuclei, provide a straightforward route to large, diffraction quality crystals. While simple in principle, seeding is difficult in practice because the experimental procedure requires the crystallographer to disrupt the equilibrium of the crystallization droplet, and oftentimes ruining the crystallization experiment. This difficulty often leaves seeding as a ‘last resort’ technique. Another technique, in meso crystallization, maintains membrane proteins in a native-like membrane throughout the process of crystallization and has yielded very high quality crystals and structures of previously intractable membrane proteins. Unfortunately, the in meso method requires handling highly viscous lipid phases with specialized mixing and dispensing tools, and is thus limited to dedicated crystallographers and labs with robotic formulation systems. Regardless of the crystallization technique used, membrane protein crystals are incredibly fragile, so the final step of harvesting and mounting crystals prior to X-ray diffraction also hampers progress in structural studies. This dissertation details the development and application of a suite of microfluidic crystallization platforms designed to overcome technical difficulties in membrane protein crystallization. Specifically, these platforms enable crystallization condition screening for either seeding techniques or in meso techniques and subsequent in situ X-ray crystallography. This work improves upon the construction of X-ray transparent devices previously designed in the Kenis group and applies them to membrane protein crystallography. In Chapter 2, devices for separating nucleation and growth via crystal seeding were developed and applied to a model soluble protein and a target membrane protein. In Chapter 3, a novel microfluidic method for formulating in meso crystallization trials was developed and used to crystallize and solve the structure of a membrane protein. In Chapter 4, in meso crystallization devices for high-throughput screening and optimization experiments were designed, and crystallization conditions for several membrane proteins of unknown structure were discovered. In the interest of directly studying protein-ligand or protein-drug interactions, a novel microfluidic method for growing and subsequently soaking crystals in meso was developed and applied to a model crystallization system in Chapter 5. In summary, this work details the development of microfluidic platforms that automate membrane protein crystallization through a variety of techniques. These devices incorporate fine control at the nanoliter scale and in situ analysis into high-throughput arrays to facilitate membrane protein structure determination. The development of platforms for in meso crystallization is particularly significant, as they represent the first X-ray transparent microfluidic platforms for in meso crystallization which also push the limits of scale and throughput when compared to state-of-the-art robotic in meso techniques. Further, when extended to studying protein-ligand and protein-drug systems via soaking, the in meso approach demonstrated here presents an attractive route to develop and study pharmaceuticals

Mesophase-based approaches for on-chip membrane protein crystallization and structure determination

Transmembrane proteins traverse the lipid bilayers of cell membranes and play a highly important role in many processes in vivo. Malfunctions of membrane proteins have been shown to cause a variety of disease states, for example, cystic fibrosis, hereditary hearing loss, and hypothyroidism. Furthermore, membrane proteins are targets of over 60% of all drugs available on the market. Thus, information on membrane protein function is of immense importance for the understanding of processes associated with disease states and for the development of new and improved therapeutics. Detailed spatial structures of proteins are typically obtained from crystal X-ray diffraction data. Crystallizing membrane proteins, however, is extremely difficult due to their amphiphilic properties that affect their stability in aqueous solutions. The trial-and-error nature of the crystallization process requires large-scale screening efforts, which is often hampered by the limited availability of membrane protein samples. In meso crystallization is a powerful alternative to the traditional crystallization of membrane proteins directly from aqueous solutions. The method involves reconstitution of the proteins into so-called lipidic mesophases that are comprised of lipid bilayers, providing a native-like environment for the proteins. Lipidic mesophases form spontaneously upon mixing of the aqueous solution of a protein with a lipid. The microstructural properties of the mesophase play a highly important role in crystallogenesis in meso and depend on the composition of the overall mixture in a complex manner. Understanding the effect of mesophase microstructure on the outcome of crystallization trials is necessary for improving the success rate of the process. Mesophases, however, are difficult to handle due to their high viscosity, and require special mixing and dispensing tools both for protein crystallization and for microstructural studies. The properties of lipidic mesophases also hampered miniaturization using microfluidic technologies, commonly used for screening of protein crystallization from solutions. Microfluidic platforms developed in this dissertation are the only examples of microfluidic devices that combine mesophase-handling capabilities and X-ray transparency as required for in situ analysis of mesophases and of protein crystals. The platforms provide a route to reduce the preparative scale, automate sample formulation, and eliminate manual handling in two distinct aspects of mesophase-based technologies: (i) screening of the structure/composition relationships of the properties of lipidic mesophases, and (ii) in meso crystallization of membrane proteins. Macroscale studies of the phase behavior of lipidic mesophases with additives typically present in membrane protein crystallization highlight the complexity of structure/composition relationships in these systems, as discussed in Chapter 2. These studies are highly laborious and require the preparation of a large number of samples with systematically varying compositions. Microfluidic platforms developed and validated in Chapters 3 and 4 allow for automated simultaneous formulation of multiple samples and scale down the amount of material per sample at least 300-fold compared to the standard macroscale method. X-ray transparency of the platforms enables small-angle X-ray scattering analysis on-chip, as required to establish the microstructure of the mesophases. The platforms address two types of structural studies of mesophases. The microfluidic system presented in Chapter 3 is designed for the studies of the effect of additives on the microstructure of mesophases in multicomponent crystallization mixtures. The chip developed in Chapter 4 is applicable for studies of phase behavior in binary lipid/water mixtures, which is necessary for the understanding of fundamental principles of self-assembly in lipidic systems and for assessing suitability of novel lipids for in meso crystallization. Chapter 5 describes a microfluidic platform for in meso crystallization of membrane proteins. The platform reduces the amount of material per trial 7-fold compared to similar macroscale methods. Platform architecture has been validated by crystallizing a membrane protein Photosynthetic Reaction Center (RC) from Rhodobacter Sphaeroides. Furthermore, crystal structure of RC was solved using X-ray diffraction data collected on-chip. Thus, the platform fully eliminates manual crystal harvesting and is a highly promising tool for structural biology. The platform is uniquely positioned for the simultaneous analysis of the protein crystals and the surrounding mesophase, which is not possible with existing macroscale approaches. This information is invaluable for unraveling the factors defining the outcome of crystallization trials and for improving the success rate in membrane protein crystallization

A Method for the Structural Investigation of Membrane Proteins

We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time

Monoolein Lipid Phases as Incorporation and Enrichment Materials for Membrane Protein Crystallization

The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase

Design of ultra-swollen lipidic mesophases for the crystallization of membrane proteins with large extracellular domains

In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3-5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d?Pn3m?Ia3d and Pn3m?Im3m?Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology

X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

abstract: Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.The final version of this article, as published in Scientific Data, can be viewed online at: https://www.nature.com/articles/sdata20162

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology