Membrane Protein Crystallization Research Articles

Insights into Crystallization of Neuronal Nicotinic α4β2 Receptor in Polarized Lipid Matrices

Obtaining high-resolution 3D structures of membrane proteins through X-ray crystallography remains a longstanding bottleneck in the field of structural biology. This challenge has led to the optimization of purification methods to acquire high-yielding, pure proteins suitable for crystallization. In this study, we performed crystallization screenings of purified human α4β2 nAChR using a polarized in meso method. After reconstituting the detergent-solubilized α4β2 nAChR into the LCP matrix, the samples were incubated in a polarized lipid matrix using the RMP@LMx device developed in our laboratory. The results showed that under these conditions, the α4β2-nAChR-LFC 16 complex gave a mobile fraction >0.8, suggesting that its diffusion in the polarized lipid matrix is favorable for crystal nucleation. Voltages above 70 mV restricted crystal formation due to sample dehydration. Furthermore, a lipid analysis using UPLC-ESI MS/MS revealed a profile necessary for preserving protein integrity and promoting diffusion across the LCP. We harvested a single crystal and subjected it to X-ray diffraction, resulting in reflections comparable to previous studies of the muscle-type nAChR from Torpedo californica. X-ray diffraction of a single crystal gave distinct low-resolution diffractions of protein nature. These findings lay the groundwork for further optimization of membrane protein crystallization in polarized in meso phases.

PLMC: Language Model of Protein Sequences Enhances Protein Crystallization Prediction.

X-ray diffraction crystallography has been most widely used for protein three-dimensional (3D) structure determination for which whether proteins are crystallizable is a central prerequisite. Yet, there are a number of procedures during protein crystallization, including protein material production, purification, and crystal production, which take turns affecting the crystallization outcome. Due to the expensive and laborious nature of this multi-stage process, various computational tools have been developed to predict protein crystallization propensity, which is then used to guide the experimental determination. In this study, we presented a novel deep learning framework, PLMC, to improve multi-stage protein crystallization propensity prediction by leveraging a pre-trained protein language model. To effectively train PLMC, two groups of features of each protein were integrated into a more comprehensive representation, including protein language embeddings from the large-scale protein sequence database and a handcrafted feature set consisting of physicochemical, sequence-based and disordered-related information. These features were further separately embedded for refinement, and then concatenated for the final prediction. Notably, our extensive benchmarking tests demonstrate that PLMC greatly outperforms other state-of-the-art methods by achieving AUC scores of 0.773, 0.893, and 0.913, respectively, at the aforementioned individual stages, and 0.982 at the final crystallization stage. Furthermore, PLMC is shown to be superior for predicting the crystallization of both globular and membrane proteins, as demonstrated by an AUC score of 0.991 for the latter. These results suggest the significant potential of PLMC in assisting researchers with the experimental design of crystallizable protein variants.

His1-tagged DM or DDM detergent micelles are reversibly conjugated by nickel ions

Specific conjugation of decyl β-D-maltoside (DM) or dodecyl β-D-maltoside (DDM) detergent micelles is accomplished between pH 7.0–8.5 in the presence of an amphiphilic analog of the amino acid histidine, bound to a 10-carbon hydrocarbon chain (His1-C10) and Ni2+ ions. Following addition of 10–15 wt% PEG-6000 as precipitant, phase separation in the form of oil-rich globules (30–600 µm) is observed by light microscopy. Other divalent cations: Zn2+, Fe2+, Cu2+ lead to dark precipitates rather than colorless globules; while Mg2+, Ca2+ do not promote any phase separation at all. Even in the absence of precipitant, dynamic light scattering (DLS) measurements demonstrate that DM micelles (hydrodynamic size ~ 6 nm) or DDM micelles (8 nm) self-associate into larger particles (9 nm and 411 nm for DM; 10 nm and 982 nm for DDM) in the presence of His1-C10 and nickel ions. Micellar conjugation is partially reversible in the presence of water soluble 50 mM EDTA, histidine or imidazole chelators. Cryo-transmission electron microscopy (cryo-TEM) imaging revealed the formation of non-uniformly dense detergent aggregates for both DM and DDM micelles in the presence of precipitant. The possible utility of such His1-tagged DM or DDM micelles for promoting crystallization of integral membrane proteins is discussed.

Development of a novel lipid for the in meso crystallisation and structure determination of membrane proteins and the impact of 2-MAG on mesophase behaviour

Development of a novel lipid for the <i>in meso</i> crystallisation and structure determination of membrane proteins and the impact of 2-MAG on mesophase behaviour

A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals

Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.

Microdialysis on-chip crystallization of soluble and membrane proteins with the MicroCrys platform and in situ X-ray diffraction case studies

The MicroCrys platform was developed to automate on-chip dialysis crystallization of proteins via chemical composition and temperature control, facilitating the optimization of crystallization conditions and the exploration of protein phase diagrams.

Specific Xray diffraction patterns of membrane proteins caused by secondary structure collinearity

Diffraction anisotropy is a phenomenon that impacts more specifically membrane proteins, compared to soluble ones, but the reasons for this discrepancy remained unclear. Often, it is referred to a difference in resolution limits between highest and lowest diffraction limits as a signature for anisotropy. We show in this article that there is no single correlation between anisotropy and difference in resolution limits, with notably a substantial number of structures displaying various anisotropy with no difference in resolution limits. We further investigated diffraction intensity profiles, and observed a peak centred on 4.9 Å resolution more predominant in membrane proteins. Since this peak is in the region corresponding to secondary structures, we investigated the influence of secondary structure ratio. We showed that secondary structure content has little influence on this profile, while secondary structure collinearity in membrane proteins correlate with a stronger peak. Finally, we could further show that the presence of this peak is linked to higher diffraction anisotropy. These results bring to light a specific diffraction of membrane protein crystals, which calls for a specific handling by crystallographic software. It also brings an explanation for investigators struggling with their anisotropic data.

Nanoporous Membranes of Densely Packed Carbon Nanotubes Formed by Lipid-Mediated Self-Assembly.

Nanofiltration technology faces the competing challenges of achieving high fluid flux through uniformly narrow pores of a mechanically and chemically stable filter. Supported dense-packed 2D-crystals of single-walled carbon nanotube (CNT) porins with ∼1 nm wide pores could, in principle, meet these challenges. However, such CNT membranes cannot currently be synthesized at high pore density. Here, we use computer simulations to explore lipid-mediated self-assembly as a route toward densely packed CNT membranes, motivated by the analogy to membrane-protein 2D crystallization. In large-scale coarse-grained molecular dynamics (MD) simulations, we find that CNTs in lipid membranes readily self-assemble into large clusters. Lipids trapped between the CNTs lubricate CNT repacking upon collisions of diffusing clusters, thereby facilitating the formation of large ordered structures. Cluster diffusion follows the Saffman-Delbrück law and its generalization by Hughes, Pailthorpe, and White. On longer time scales, we expect the formation of close-packed CNT structures by depletion of the intervening shared annular lipid shell, depending on the relative strength of CNT-CNT and CNT-lipid interactions. Our simulations identify CNT length, diameter, and end functionalization as major factors for the self-assembly of CNT membranes.

Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase

Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP.

Surfactant-Mediated Solubilization of Myelin Figures: A Multistep Morphological Cascade.

Lamellar mesophases of insoluble lipids are readily solubilized by the micellar mesophases of soluble surfactants. This simple process underscores a broad array of biochemical methodologies, including purification, reconstitution, and crystallization of membrane proteins, as well as the isolation of detergent-resistant membrane fractions. Although much is now known about the thermodynamic driving forces of membrane solubilization, the kinetic pathways by which the surfactant alters vesicular mesophases are only beginning to be appreciated. Little is known about how these interactions affect the solubilization of more complex, multilamellar mesophases. Here, we investigate how a common zwitterionic detergent affects the solubilization of a smectic, multilamellar, cylindrical mesophase of lipids, called the myelin figure. Our results reveal that myelin solubilization occurs in a multistep manner, producing a well-defined sequence of morphologically distinct intermediates en route to complete solubilization. The kinetic processes producing these intermediates include (1) coiling, which encompasses the formation, propagation, and tightening of extended helices; (2) thinning, which reflects the unbinding of lamellae in the smectic stacks; and (3) detachment or retraction, which either dissociates the myelinic protrusion from the source lipid mass or returns the myelinic protrusion to the source lipid mass─all in transit toward complete solubilization. These occasionally overlapping steps are most pronounced in single-lipid component myelins, while compositionally graded multicomponent myelins inhibit the coiling step and detach more frequently. Taken together, the appearance of these intermediates during the solubilization of myelins suggests a complex free-energy landscape characterizing myelin solubilization populated by metastable, morphological intermediates correlated with locally minimized changes in energy dependent upon the mesophase's composition. This then predicts the accessibility of structurally distinct, kinetic intermediates─such as loose and tight coiled helices, peeled myelins, retracted tubes, and detached protrusions─before reaching the stable ground state corresponding to a dissolved suspension of mixed surfactant-lipid micelles.

Mechanisms of membrane protein crystallization in ‘bicelles’

Despite remarkable progress, mainly due to the development of LCP and ‘bicelle’ crystallization, lack of structural information remains a bottleneck in membrane protein (MP) research. A major reason is the absence of complete understanding of the mechanism of crystallization. Here we present small-angle scattering studies of the evolution of the “bicelle” crystallization matrix in the course of MP crystal growth. Initially, the matrix corresponds to liquid-like bicelle state. However, after adding the precipitant, the crystallization matrix transforms to jelly-like state. The data suggest that this final phase is composed of interconnected ribbon-like bilayers, where crystals grow. A small amount of multilamellar phase appears, and its volume increases concomitantly with the volume of growing crystals. We suggest that the lamellar phase surrounds the crystals and is critical for crystal growth, which is also common for LCP crystallization. The study discloses mechanisms of “bicelle” MP crystallization and will support rational design of crystallization.

Structural Basis of Agonist Capture by Regulatory C1 Domain of PKC

Diacylglycerol (DAG)‐sensing C1 domains of Protein kinase C (PKC) isoforms are targets for therapeutic intervention in cancers, neurodegenerative disorders, and HIV latency reversal. High‐resolution structural information on the C1 domains complexed to DAG or exogenous PKC agonists remained elusive for nearly 3 decades. This is attributed to the extreme hydrophobicity of the C1 complexes and the need to create an optimal membrane‐mimicking environment to facilitate the complex formation and crystallization. We overcame these challenges and obtained high‐resolution crystal structures of the C1B domain from PKCδ (C1Bδ) complexed to DAG and four potent PKC agonists of pharmacological relevance. The structures reveal precise stereospecific binding modes and hydrophobic interactions formed by the lipophilic groups of DAG and DAG lactone (AJH‐836) with C1Bδ. Comparison of the C1Bδ complexes with PDBu (a tumor‐promoting phorbol ester) and Prostratin (a candidate for the HIV latency reversal) provides a structural rationale for their distinct PKC‐mediated cellular responses. The complex with Ingenol mebutate, a drug clinically approved for the treatment of actinic keratosis, shows how its angelyl group completes the hydrophobic rim of C1Bδ. The analysis of all five complexes enabled us to assign the functional roles to residues that form the consensus sequence of DAG‐sensitive C1 domains. We conclude that the ligand promiscuity of C1 domains stems from a subset of conserved residues that can “cage” chemically diverse ligands while interfacing with the membrane environment. These structures serve as valuable guides for pushing the frontiers of PKC pharmacology and suggest a new strategy for the crystallization of peripheral membrane proteins.

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.

Reversible Conjugation of Non-ionic Detergent Micelles Promotes Partitioning of Membrane Proteins under Non-denaturing Conditions.

In the decades’-long quest for high-quality membrane protein (MP) crystals, non-ionic detergent micelles have primarily served as a passive shield against protein aggregation in aqueous solution and/or as a conformation stabilizing environment. We have focused on exploiting the physical chemistry of detergent micelles in order to direct intrinsic MP/detergent complexes to assemble via conjugation under ambient conditions, thereby permitting finely tuned control over the micelle cloud point. In the current work, three commercially available amphiphilic, bipyridine chelators in combination with Fe2+ or Ni2+ were tested for their ability to conjugate non-ionic detergent micelles both in the presence and absence of an encapsulated bacteriorhodopsin molecule. Water-soluble chelators were added, and results were monitored with light microscopy and dynamic light scattering (DLS). [Bipyridine:metal] complexes produced micellar conjugates, which appeared as oil-rich globules (10–200 μm) under a light microscope. DLS analysis demonstrated that micellar conjugation is complete 20 min after the introduction of the amphiphilic complex, and that the conjugation process can be fully or partially reversed with water-soluble chelators. This process of controlled conjugation/deconjugation under nondenaturing conditions provides broader flexibility in the choice of detergent for intrinsic MP purification and conformational flexibility during the crystallization procedure.

MicroED: conception, practice and future opportunities.

This article documents a keynote seminar presented at the IUCr Congress in Prague, 2021. The cryo-EM method microcrystal electron diffraction is described and put in the context of macromolecular electron crystallography from its origins in 2D crystals of membrane proteins to today's application to 3D crystals a millionth the size of that needed for X-ray crystallography. Milestones in method development and applications are described with an outlook to the future.

Stabilization and structure determination of integral membrane proteins by termini restraining.

Integral membrane proteins isolated from cellular environment often lose activity and native conformation required for functional analyses and structural studies. Even in their native state, they lack sufficient surfaces to form crystal contacts. Furthermore, most of them are too small for cryogenic electron microscopy detection and too big for solution NMR. To overcome these difficulties, we recently developed a strategy to stabilize the folded state of membrane proteins by restraining their two termini with a self-assembling protein coupler. The termini-restrained membrane proteins from distinct functional families retain their activities and show increased stability and yield. This strategy enables their structure determination at near-atomic resolution by facilitating the entire pipeline from crystallization, crystal identification, diffraction enhancement and phase determination, to electron density improvement. Furthermore, stabilization of membrane proteins enables their biochemical and biophysical characterization. Here we present the protocol of membrane protein engineering (2 weeks), quality assessment (1-2 weeks), protein production (1-6 weeks), crystallization (1-2 weeks), diffraction improvement (1-3 months) and crystallographic data analysis (1 week). This protocol is intended not only for structural biologists, but also for biochemists, biophysicists and pharmaceutical scientists whose research focuses on membrane proteins.

A solution NMR view of lipidic cubic phases: Structure, dynamics, and beyond

Nuclear magnetic resonance (NMR) spectroscopy is well-established nowadays for the elucidation of the 3D structures of proteins and protein complexes, the evaluation of biomolecular dynamics with atomistic resolution across a range of time scales, the screening of drug candidates with site specificity, and for the quantitation of molecular translational diffusion. Lyotropic lipidic cubic phases (LCPs) are lipid bilayer-based materials with a complex geometry, formed via the spontaneous self-assembly of certain lipids in an aqueous environment at specific temperature ranges. LCPs have been successfully applied to the in meso crystallization of membrane proteins for structural studies by X-ray crystallography, and have also shown promising potential for serving as matrices for drug and nutrient delivery/release in vivo. The characterization of the structural and dynamics properties of LCPs is of significant interest for the application of these materials. Here we present a systematic review detailing the characterization of LCPs by solution NMR. Using LCPs formed by monoolein (MO) as an example, various aspects of LCPs readily accessible by solution NMR are covered, including spectral perturbation in the presence of additives, quantification of hydration levels, 13C relaxation-based measurements for studying atom-specific dynamics along the MO hydrocarbon chain, PGSE NMR measurement of translational diffusion and its correlation with release profiles, and the encapsulation of soluble proteins in LCPs. A brief discussion of future perspectives for the characterization of LCPs by solution NMR is also presented.

Insights into the mechanism of high lipid–detergent crystallization of membrane proteins

Obtaining well diffracting crystals of membrane proteins is often challenging, but chances can be improved by crystallizing them in lipidic conditions that mimic their natural membrane environments. One approach is the high lipid–detergent (HiLiDe) method, which works by mixing the target protein with high concentrations of lipid and detergent prior to crystallization. Although this approach is convenient and flexible, understanding the effects of systematically varying lipid/detergent ratios and a characterization of the lipid phases that form during crystallization would be useful. Here, a HiLiDe phase diagram is reported for the model membrane protein MhsT, which tracks the precipitation and crystallization zones as a function of lipid and detergent concentrations, and is augmented with data on crystal sizes and diffraction properties. Additionally, the crystallization of SERCA1a solubilized directly with native lipids is characterized as a function of detergent concentration. Finally, HiLiDe crystallization drops are analysed with transmission electron microscopy, which among other features reveals liposomes, stacked lamellae that may represent crystal precursors, and mature crystals with clearly discernible packing arrangements. The results emphasize the significance of optimizing lipid/detergent ratios over broad ranges and provide insights into the mechanism of HiLiDe crystallization.

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown into highly ordered two-dimensional (2D) crystals under favorable conditions. The quality of the grown 2D crystals is crucial to the resolution of the final reconstruction via 2D image processing. Over the years, lipid monolayers have been used as a supporting layer to foster the 2D crystallization of peripheral membrane proteins as well as soluble proteins. This method can also be applied to 2D crystallization of integral membrane proteins but requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the monolayer. A lipid monolayer forms at the air-water interface such that the polar lipid head groups remain hydrated in the aqueous phase and the non-polar, acyl chains, tails partition into the air, breaking the surface tension and flattening the water surface. The charged nature or distinctive chemical moieties of the head groups provide affinity for proteins in solution, promoting binding for 2D array formation. A newly formed monolayer with the 2D array can be readily transfer into an electron microscope (EM) on a carbon-coated copper grid used to lift and support the crystalline array. In this work, we describe a lipid monolayer methodology for cryogenic electron microscopic (cryo-EM) imaging.

NMR spectroscopy of lipidic cubic phases.

Lipidic cubic phase (LCP) structures have been used for stabilisation and crystallisation of membrane proteins and show promising properties as drug carriers. In this mini-review, we present how NMR spectroscopy has played a major role in understanding the physico-chemical properties of LCPs and how recent advances in pulsed field gradient NMR techniques open new perspectives in characterising encapsulated molecules.