Membrane Protein Crystallography Research Articles

Mix-and-extrude: high-viscosity sample injection towards time-resolved protein crystallography.

Time-resolved crystallography enables the visualization of protein molecular motion during a reaction. Although light is often used to initiate reactions in time-resolved crystallography, only a small number of proteins can be activated by light. However, many biological reactions can be triggered by the interaction between proteins and ligands. The sample delivery method presented here uses a mix-and-extrude approach based on 3D-printed microchannels in conjunction with a micronozzle. The diffusive mixing enables the study of the dynamics of samples in viscous media. The device design allows mixing of the ligands and protein crystals in 2 to 20 s. The device characterization using a model system (fluorescence quenching of iq-mEmerald proteins by copper ions) demonstrated that ligand and protein crystals, each within lipidic cubic phase, can be mixed efficiently. The potential of this approach for time-resolved membrane protein crystallography to support the development of new drugs is discussed.

Lipid doping of the sponge (L3) mesophase.

The polymorphism of lipid aggregates has long attracted detailed study due to the myriad factors that determine the final mesophase observed. This study is driven by the need to understand mesophase behaviour for a number of applications, such as drug delivery and membrane protein crystallography. In the case of the latter, the role of the so-called 'sponge' (L3) mesophase has been often noted, but not extensively studied by itself. The L3 mesophase can be formed in monoolein/water systems on the addition of butanediol to water, which partitions the headgroup region of the membrane, and decreases its elastic moduli. Like cubic mesophases, it is bicontinuous, but unlike them, has no long-range translational symmetry. In our present study, we show that the formation of the L3 phase can delicately depend on the addition of dopant lipids to the mesophase. While electrostatically neutral molecules similar in shape to monoolein (DOPE, cholesterol) have little effect on the general mesophase behaviour, others (DOPC, DDM) significantly reduce the composition at which it can form. Additionally, we show that by combining cholesterol with the anionic lipid DOPG, it is possible to form the largest stable L3 mesophases observed to date, with characteristic lengths over 220 Å.

Observations of phase changes in monoolein during high viscous injection.

Serial crystallography of membrane proteins often employs high-viscosity injectors (HVIs) to deliver micrometre-sized crystals to the X-ray beam. Typically, the carrier medium is a lipidic cubic phase (LCP) media, which can also be used to nucleate and grow the crystals. However, despite the fact that the LCP is widely used with HVIs, the potential impact of the injection process on the LCP structure has not been reported and hence is not yet well understood. The self-assembled structure of the LCP can be affected by pressure, dehydration and temperature changes, all of which occur during continuous flow injection. These changes to the LCP structure may in turn impact the results of X-ray diffraction measurements from membrane protein crystals. To investigate the influence of HVIs on the structure of the LCP we conducted a study of the phase changes in monoolein/water and monoolein/buffer mixtures during continuous flow injection, at both atmospheric pressure and under vacuum. The reservoir pressure in the HVI was tracked to determine if there is any correlation with the phase behaviour of the LCP. The results indicated that, even though the reservoir pressure underwent (at times) significant variation, this did not appear to correlate with observed phase changes in the sample stream or correspond to shifts in the LCP lattice parameter. During vacuum injection, there was a three-way coexistence of the gyroid cubic phase, diamond cubic phase and lamellar phase. During injection at atmospheric pressure, the coexistence of a cubic phase and lamellar phase in the monoolein/water mixtures was also observed. The degree to which the lamellar phase is formed was found to be strongly dependent on the co-flowing gas conditions used to stabilize the LCP stream. A combination of laboratory-based optical polarization microscopy and simulation studies was used to investigate these observations.

Membrane protein crystallography in the era of modern structural biology.

The aim of structural biology has been always the study of biological macromolecules structures and their mechanistic behaviour at molecular level. To achieve its goal, multiple biophysical methods and approaches have become part of the structural biology toolbox. Considered as one of the pillars of structural biology, X-ray crystallography has been the most successful method for solving three-dimensional protein structures at atomic level to date. It is however limited by the success in obtaining well-ordered protein crystals that diffract at high resolution. This is especially true for challenging targets such as membrane proteins (MPs). Understanding structure-function relationships of MPs at the biochemical level is vital for medicine and drug discovery as they play critical roles in many cellular processes. Though difficult, structure determination of MPs by X-ray crystallography has significantly improved in the last two decades, mainly due to many relevant technological and methodological developments. Today, numerous MP crystal structures have been solved, revealing many of their mechanisms of action. Yet the field of structural biology has also been through significant technological breakthroughs in recent years, particularly in the fields of single particle electron microscopy (cryo-EM) and X-ray free electron lasers (XFELs). Here we summarise the most important advancements in the field of MP crystallography and the significance of these developments in the present era of modern structural biology.

Harnessing the power of an X-ray laser for serial crystallography of membrane proteins crystallized in lipidic cubic phase.

Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution is determined and compared with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, the capability of utilizing high XFEL beam transmissions is demonstrated, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete dataset. The experimental setup presented herein can be applied to future SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.

Usefulness of oils for cleaning the host matrix and for cryoprotection of lipidic cubic phase crystals

The lipidic cubic phase method is an effective approach for membrane protein crystallography. The in meso grown crystals are usually cryocooled directly without removing the host matrix from the harvested crystal surface. However, the host matrix often causes the appearance of scattering rings and an increase in background scattering during the data collection. Moreover, the frozen host matrix sometimes becomes opaque and it can hinder conventional crystal centering. In this study, several oils were examined for their ability to clean the host matrix and to provide cryoprotection for crystals grown in the lipidic cubic phase. Several of the tested oils appeared to be useful in terms of their effect on crystal stability and background scattering. This method should be of value for the collection of highly accurate data sets.

Generation of Conformation-Specific Antibody Fragments for Crystallization of the Multidrug Resistance Transporter MdfA.

A major hurdle in membrane protein crystallography is generating crystals diffracting sufficiently for structure determination. This is often attributed not only to the difficulty of obtaining functionally active protein in mg amounts but also to the intrinsic flexibility of its multiple conformations. The cocrystallization of membrane proteins with antibody fragments has been reported as an effective approach to improve the diffraction quality of membrane protein crystals by limiting the intrinsic flexibility. Isolating suitable antibody fragments recognizing a single conformation of a native membrane protein is not a straightforward task. However, by a systematic screening approach, the time to obtain suitable antibody fragments and consequently the chance of obtaining diffracting crystals can be reduced. In this chapter, we describe a protocol for the generation of Fab fragments recognizing the native conformation of a major facilitator superfamily (MFS)-type MDR transporter MdfA from Escherichia coli. We confirmed that the use of Fab fragments was efficient for stabilization of MdfA and improvement of its crystallization properties.

Screening and Identifying Membrane Proteins Favorable for Crystallization.

This unit addresses several critical challenges associated with membrane protein crystallography by screening membrane proteins from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebral tissue for biochemical properties favorable for crystallization. First, a tissue sample or cell pellet is obtained. The cells are isolated, washed, and then lysed either by sonication, bead beating, or manual homogenization. Membrane proteins are fractionated from the lysates by centrifugation and solubilized in a mild detergent suitable for crystallization, such as n-dodecyl-β-maltoside (DDM). Detergent extracts are then centrifuged, heat precipitated, and filtered to remove insoluble, thermally unstable, and/or aggregated proteins. Samples are then prepared for analysis by mass spectrometry: proteins are precipitated by methanol/chloroform extraction and subjected to reduction, alkylation, and protease digestion. The resulting peptides are passed through a detergent removal column, desalted, rehydrated in 0.1% formic acid (v/v), and identified by LC-MS/MS. © 2017 by John Wiley & Sons, Inc.

Lipid-like Peptides can Stabilize Integral Membrane Proteins for Biophysical and Structural Studies.

A crucial bottleneck in membrane protein structural biology is the difficulty in identifying a detergent that can maintain the stability and functionality of integral membrane proteins (IMPs). Detergents are poor membrane mimics, and their common use in membrane protein crystallography may be one reason for the challenges in obtaining high‐resolution crystal structures of many IMP families. Lipid‐like peptides (LLPs) have detergent‐like properties and have been proposed as alternatives for the solubilization of G protein‐coupled receptors and other membrane proteins. Here, we systematically analyzed the stabilizing effect of LLPs on integral membrane proteins of different families. We found that LLPs could significantly stabilize detergent‐solubilized IMPs in vitro. This stabilizing effect depended on the chemical nature of the LLP and the intrinsic stability of a particular IMP in the detergent. Our results suggest that screening a subset of LLPs is sufficient to stabilize a particular IMP, which can have a substantial impact on the crystallization and quality of the crystal.

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment-mediated crystallography.

Fv antibody fragments have been used as co-crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large-scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (VL ) domain, an intervening removable affinity tag (iRAT), and a variable heavy (VH ) domain is expressed by a Gram-positive bacterial secretion system. This method ensures stoichiometric expression of VL and VH from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site-specific protease during the purification process to yield tag-free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT-based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA-approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies.

Advances in in situ X-ray Crystallography of Membrane Proteins

In the last decade, crystallisation of membrane proteins in lipidic cubic phases (LCP; in meso) boosted the numbers and resolution of membrane-protein structures. However, membrane-protein crystals are usually small and notoriously difficult to harvest from the highly viscous LCP. Recently, an in situ in meso approach has been introduced, in which crystals are not harvested and flash-frozen but placed in the X-ray beam within the mesophase at room temperature. In this study, we introduce novel in situ in meso plates that show significantly less background scattering, are easier to handle, and are variable with respect to the crystallisation volume. Moreover, we developed holders for either individual or up to four in situ wells at a time, which are easy and cheap to produce, easy to handle, reusable, and compatible with measurements at room temperature and under cryogenic conditions. Because the holders are attached to standard ALS-type goniometer bases, they allow for storage and shipping of entire wells (with typically several dozens of crystals) in Universal V1-Pucks under liquid nitrogen and for auto-mounting at synchrotrons. We validated the new setups using water-soluble hen egg lysozyme and the membrane protein bacteriorhodopsin. In conclusion, this study demonstrates the potential of combining in meso crystallisation with in situ diffraction and the possibility to store, ship, and measure crystals under cryogenic conditions for obtaining structural information on membrane proteins. In conjunction with the current developments at synchrotrons like smaller beams, faster detectors, and software for multi-crystal strategies, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.

Crystal Dehydration in Membrane Protein Crystallography.

Crystal dehydration has been successfully implemented to facilitate the structural solution of a number of soluble and membrane protein structures over the years. This chapter will present the currently available tools to undertake controlled crystal dehydration, focusing on some successful membrane protein cases. Also discussed here will be some practical considerations regarding membrane protein crystals and the relationship between different techniques in order to help researchers to select the most suitable technique for their projects.

Exploiting Microbeams for Membrane Protein Structure Determination.

A reproducible, and sample independent means of predictably obtaining large, well-ordered crystals has proven elusive in macromolecular crystallography. In the structure determination pipeline, crystallisation often proves to be a rate-limiting step, and the process of obtaining even small or badly ordered crystals can prove time-consuming and laborious. This is particularly true in the field of membrane protein crystallography and this is reflected in the limited number of unique membrane protein structures deposited in the protein data bank (less than 650 by June 2016 – http://blanco.biomol.uci.edu/mpstruc). Over recent years the requirement for, and time and cost associated with obtaining, large crystals has been partially alleviated through the development of beamline instrumentation allowing data collection, and structure solution, from ever-smaller crystals. Advances in several areas have led to a step change in what might be considered achievable during a synchrotron trip over the last decade. This chapter will briefly review the current status of the field, the tools available to ease data collection and processing, and give some examples of exploitation of these for membrane protein microfocus macromolecular crystallography.

Large-scale identification of membrane proteins with properties favorable for crystallization.

Membrane protein crystallography is notoriously difficult due to challenges in protein expression and issues of degradation and structural stability. We have developed a novel method for large-scale screening of native sources for integral membrane proteins that have intrinsic biochemical properties favorable for crystallization. Highly expressed membrane proteins that are thermally stable and nonaggregating in detergent solutions were identified by mass spectrometry from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebrum. Many of the membrane proteins identified had been crystallized previously, supporting the promise of the approach. Most identified proteins have known functions and include high-value targets such as transporters and ATPases. To validate the method, we recombinantly expressed and purified the yeast protein, Yop1, which is responsible for endoplasmic reticulum curvature. We demonstrate that Yop1 can be purified with the detergent dodecylmaltoside without aggregating.

Two-dimensional membrane protein crystallography at X-FELs

Two-dimensional membrane protein crystallography at X-FELs

Building Synthetic Sterols Computationally – Unlocking the Secrets of Evolution?

Cholesterol is vital in regulating the physical properties of animal cell membranes. While it remains unclear what renders cholesterol so unique, it is known that other sterols are less capable in modulating membrane properties, and there are membrane proteins whose function is dependent on cholesterol. Practical applications of cholesterol include its use in liposomes in drug delivery and cosmetics, cholesterol-based detergents in membrane protein crystallography, its fluorescent analogs in studies of cholesterol transport in cells and tissues, etc. Clearly, in spite of their difficult synthesis, producing the synthetic analogs of cholesterol is of great commercial and scientific interest. In this article, we discuss how synthetic sterols non-existent in nature can be used to elucidate the roles of cholesterol’s structural elements. To this end, we discuss recent atomistic molecular dynamics simulation studies that have predicted new synthetic sterols with properties comparable to those of cholesterol. We also discuss more recent experimental studies that have vindicated these predictions. The paper highlights the strength of computational simulations in making predictions for synthetic biology, thereby guiding experiments.

Advances in membrane protein crystallography: in situ and in meso data collection.

Advances in membrane protein crystallography: in situ and in meso data collection.

Hydrogen bond network around the semiquinone of the secondary quinone acceptor Q(B) in bacterial photosynthetic reaction centers.

By utilizing a combined pulsed EPR and DFT approach, the high-resolution structure of the QB site semiquinone (SQB) was determined. The development of such a technique is crucial toward an understanding of protein-bound semiquinones on the structural level, as (i) membrane protein crystallography typically results in low resolution structures, and (ii) obtaining protein crystals in the semiquinone form is rarely feasible. The SQB hydrogen bond network was investigated with Q- (∼34 GHz) and X-band (∼9.7 GHz) pulsed EPR spectroscopy on fully deuterated reactions centers from Rhodobacter sphaeroides. Simulations in the SQB g-tensor reference frame provided the principal values and directions of the H-bond proton hyperfine tensors. Three protons were detected, one with an anisotropic tensor component, T = 4.6 MHz, assigned to the histidine NδH of His-L190, and two others with similar anisotropic constants T = 3.2 and 3.0 MHz assigned to the peptide NpH of Gly-L225 and Ile-L224, respectively. Despite the strong similarity in the peptide couplings, all hyperfine tensors were resolved in the Q-band ENDOR spectra. The Euler angles describing the series of rotations that bring the hyperfine tensors into the SQB g-tensor reference frame were obtained by least-squares fitting of the spectral simulations to the ENDOR data. These Euler angles show the locations of the hydrogen bonded protons with respect to the semiquinone. Our geometry optimized model of SQB used in previous DFT work is in strong agreement with the angular constraints from the spectral simulations, providing the foundation for future joint pulsed EPR and DFT semiquinone structural determinations in other proteins.

Activation of corticotropin-releasing factor 1 receptor: insights from molecular dynamics simulations.

G-protein-coupled receptors (GPCRs) constitute the largest family of membrane-bound proteins involved in translation of extracellular signals into intracellular responses. They regulate diverse physiological and pathophysiological processes, and hence, they are prime drug targets for therapeutic intervention. In spite of the recent advancements in membrane protein crystallography, limited information is available on the molecular signatures of activation of GPCRs. Although few studies have been reported for class A GPCRs, the activation mechanism of class B GPCRs remains unexplored. Corticotropin-releasing factor 1 receptor (CRF1R), a class B GPCR, is associated with various disease conditions including stress, anxiety, and irritable bowel syndrome. Here, we report the activation of CRF1R using accelerated molecular dynamics simulations of the apo receptor. The breakage of His155(2.50)-Glu209(3.50) and Glu209(3.50)-Thr316(6.42) interactions is found to be crucial in transition of the receptor to its active conformation. Compared to the inactive crystal structure, major structural rearrangements occurred in the intracellular region of the transmembrane (TM) domain upon activation: TM3 twisted away from TM2, and an opening of the G-protein binding site occurred as a result of the outward movements of TM5 and TM6 from the helical bundle. Further, an inward tilt of TM7 toward the helical core is observed at the extracellular side, in agreement with recent findings (Coin et al. Cell 2013, 155, 1258-1269), where it is proposed that this movement helps in establishing favorable interactions with peptide agonist. Moreover, different allosteric pathways in the inactive and active states are identified using the correlations in torsion angle space. The inactive state is found to be less dynamic as compared to the putative active state of the receptor. Results from the current study could present a model for class B GPCRs activation and aid in the design of CRF1R modulators against brain and metabolic disorders.

Quantification of detergent using colorimetric methods in membrane protein crystallography.

Membrane protein crystallography has the potential to greatly aid our understanding of membrane protein biology. Yet, membrane protein crystals remain challenging to produce. Although robust methods for the expression and purification of membrane proteins continue to be developed, the detergent component of membrane protein samples is equally important to crystallization efforts. This chapter describes the development of three colorimetric assays for the quantitation of detergent in membrane protein samples and provides detailed protocols. All of these techniques use small sample volumes and have potential applications in crystallography. The application of these techniques in crystallization prescreening, detergent concentration modification, and detergent exchange experiments is demonstrated. It has been observed that the concentration of detergent in a membrane protein sample can be just as important as the protein concentration when attempting to reproduce crystallization lead conditions.