Use of detergents in two-dimensional crystallization of membrane proteins

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.     

Two-dimensional crystallization of the ryanodine receptor Ca2+ release channel on lipid membranes

The ryanodine receptor (RyR) is the largest known membrane protein with a total molecular mass of 2.3 x 10(3) kDa. Well ordered, two-dimensional (2D) crystals are an essential prerequisite to enable RyR structure determination by electron crystallography. Conventionally, the 2D crystallization of membrane proteins is based on a 'trial-and-error' strategy, which is both time-consuming and chance-directed. By adopting a new strategy that utilizes protein sequence information and predicted transmembrane topology, we successfully crystallized the RyR on positively charged lipid membranes. Image processing of negatively stained crystals reveals that they are well ordered, with diffraction spots of IQ < or = 4 extending to approximately 20 angstroms, the resolution attainable in negative stain. The RyR crystals obtained on the charged lipid membrane have characteristics consistent with 2D arrays that have been observed in native sarcoplasmic reticulum of muscle tissues. These crystals provide ideal materials to enable structural analysis of RyR by high-resolution electron crystallography. Moreover, the reconstituted native-like 2D array provides an ideal model system to gain structural insights into the mechanism of RyR-mediated Ca2+ signaling processes, in which the intrinsic ability of RyR oligomers to organize into a 2D array plays a crucial role.     

Structure determination of secondary transport proteins by electron crystallography: two-dimensional crystallization of the betaine uptake system BetP

Structure determination at high resolution is still a challenge for membrane proteins in general, but in particular for secondary transporters due to their highly dynamic nature. X-ray structures of ten secondary transporters have recently been determined, but a thorough understanding of transport mechanisms necessitates structures at different functional states. Electron cryo-microscopy of two-dimensional (2D) crystals offers an alternative to obtain structural information at intermediate resolution. Electron crystallography is a sophisticated way to study proteins in a natural membrane environment and to track conformational changes in situ. Furthermore, basic interactions between protein and lipids can be investigated. Projection and 3-dimensional maps of six secondary transporters from different families have been determined by electron crystallography of 2D crystals at a resolution of 8 A and better. In this review, we give an overview about the principles of 2D crystallization, in particular of secondary transporters, and summarize the important steps successfully applied to establish and improve the 2D crystallization of the high-affinity glycine betaine uptake system from Corynebacterium glutamicum, BetP.     

Single particle 3D reconstruction for 2D crystal images of membrane proteins

In cases where ultra-flat cryo-preparations of well-ordered two-dimensional (2D) crystals are available, electron crystallography is a powerful method for the determination of the high-resolution structures of membrane and soluble proteins. However, crystal unbending and Fourier-filtering methods in electron crystallography three-dimensional (3D) image processing are generally limited in their performance for 2D crystals that are badly ordered or non-flat. Here we present a single particle image processing approach, which is implemented as an extension of the 2D crystallographic pipeline realized in the 2dx software package, for the determination of high-resolution 3D structures of membrane proteins. The algorithm presented, addresses the low single-to-noise ratio (SNR) of 2D crystal images by exploiting neighborhood correlation between adjacent proteins in the 2D crystal. Compared with conventional single particle processing for randomly oriented particles, the computational costs are greatly reduced due to the crystal-induced limited search space, which allows a much finer search space compared to classical single particle processing. To reduce the considerable computational costs, our software features a hybrid parallelization scheme for multi-CPU clusters and computer with high-end graphic processing units (GPUs). We successfully apply the new refinement method to the structure of the potassium channel MloK1. The calculated 3D reconstruction shows more structural details and contains less noise than the map obtained by conventional Fourier-filtering based processing of the same 2D crystal images.     

Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins

A great interest exists in producing and/or improving two-dimensional (2D) crystals of membrane proteins amenable to structural analysis by electron crystallography. Here we report on the use of the detergent n-octyl beta-d-thioglucopyranoside in 2D crystallization trials of membrane proteins with radically different structures including FhuA from the outer membrane of Escherichia coli, light-harvesting complex II from Rubrivivax gelatinosus, and Photosystem I from cyanobacterium Synechococcus sp. We have analyzed by electron microscopy the structures reconstituted after detergent removal from lipid-detergent or lipid-protein-detergent micellar solutions containing either only n-octyl beta-d-thioglucopyranoside or n-octyl beta-d-thioglucopyranoside in combination with other detergents commonly used in membrane protein biochemistry. This allowed the definition of experimental conditions in which the use of n-octyl beta-d-thioglucopyranoside could induce a considerable increase in the size of reconstituted membrane structures, up to several micrometers. An other important feature was that, in addition to reconstitution of membrane proteins into large bilayered structures, this thioglycosylated detergent also was revealed to be efficient in crystallization trials, allowing the proteins to be analyzed in large coherent two-dimensional arrays. Thus, inclusion of n-octyl beta-d-thioglucopyranoside in 2D crystallization trials appears to be a promising method for the production of large and coherent 2D crystals that will be valuable for structural analysis by electron crystallography and atomic force microscopy.     

A helical processing pipeline for EM structure determination of membrane proteins

Electron crystallography plays a key role in the structural biology of integral membrane proteins (IMPs) by offering one of the most direct means of providing insight into the functional state of these molecular machines in their lipid-associated forms, and also has the potential to facilitate examination of physiologically relevant transitional states and complexes. Helical or tubular crystals, which are the natural product of proteins crystallizing on the surface of a cylindrical vesicle, offer some unique advantages, such as three-dimensional (3D) information from a single view, compared to other crystalline forms. While a number of software packages are available for processing images of helical crystals to produce 3D electron density maps, widespread exploitation of helical image reconstruction is limited by a lack of standardized approaches and the initial effort and specialized expertise required. Our goal is to develop an integrated pipeline to enable structure determination by transmission electron microscopy (TEM) of IMPs in the form of tubular crystals. We describe here the integration of standard Fourier-Bessel helical analysis techniques into Appion, an integrated, database-driven pipeline.     

Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin

High-resolution structural data of membrane proteins can be obtained by studying 2D crystals by electron crystallography. Finding the right conditions to produce these crystals is one of the major bottlenecks encountered in 2D crystallography. Many reviews address 2D crystallization techniques in attempts to provide guidelines for crystallographers. Several techniques including new approaches to remove detergent like the biobeads technique and the development of dedicated devices have been described (dialysis and dilution machines). In addition, 2D crystallization at interfaces has been studied, the most prominent method being the 2D crystallization at the lipid monolayer. A new approach based on detergent complexation by cyclodextrins is presented in this paper. To prove the ability of cyclodextrins to remove detergent from ternary mixtures (lipid, detergent and protein) in order to get 2D crystals, this method has been tested with OmpF, a typical beta-barrel protein, and with SoPIP2;1, a typical alpha-helical protein. Experiments over different time ranges were performed to analyze the kinetic effects of detergent removal with cyclodextrins on the formation of 2D crystals. The quality of the produced crystals was assessed with negative stain electron microscopy, cryo-electron microscopy and diffraction. Both proteins yielded crystals comparable in quality to previous crystallization reports.     

Two-dimensional crystallization of membrane proteins: the lipid layer strategy

Due to the difficulty to crystallize membrane proteins, there is a considerable interest to intensify research topics aimed at developing new methods of crystallization. In this context, the lipid layer crystallization at the air/water interface, used so far for soluble proteins, has been recently adapted successfully to produce two-dimensional (2D) crystals of membrane proteins, amenable to structural analysis by electron crystallography. Besides to represent a new alternative strategy, this approach gains the advantage to decrease significantly the amount of material needed in incubation trials, thus opening the field of crystallization to those membrane proteins difficult to surexpress and/or purify. The systematic studies that have been performed on different classes of membrane proteins are reviewed and the physico-chemical processes that lead to the production of 2D crystals are addressed. The different drawbacks, advantages and perspectives of this new strategy for providing structural information on membrane proteins are discussed.     

Advances in structural and functional analysis of membrane proteins by electron crystallography

Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography.     

‘Hit and run’ serial femtosecond crystallography of a membrane kinase in the lipid cubic phase

The lipid-based bicontinuous cubic mesophase is a nanoporous membrane mimetic with applications in areas that include medicine, personal care products, foods and the basic sciences. An application of particular note concerns it use as a medium in which to grow crystals of membrane proteins for structure determination by X-ray crystallography. At least two variations of the mesophase exist. One is the highly viscous cubic phase, which has well developed long-range order. The other so-called sponge phase is considerably more fluid and lacks long-range order. The sponge phase has recently been shown to be a convenient vehicle for delivering microcrystals of membrane proteins to an X-ray free-electron laser beam for serial femtosecond crystallography (SFX). Unfortunately, the sponge phase approach calls for large amounts of protein that are not always available in the case of membrane proteins. The cubic phase offers the advantage of requiring significantly less protein for SFX but comes with its own challenges. Here, we describe the physico-chemical bases for these challenges, solutions to them and prospects for future uses of lipidic mesophases in the SFX arena.     

Membrane protein structure determination by electron crystallography

During the past year, electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer. From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope. There have also been key developments in software, and in molecular replacement and phase extension methods designed to facilitate the process of structure determination.     

Electron crystallography - the waking beauty of structural biology

Since its debut in the mid 1970s, electron crystallography has been a valuable alternative in the structure determination of biological macromolecules. Its reliance on single-layered or double-layered two-dimensionally ordered arrays and the ability to obtain structural information from small and disordered crystals make this approach particularly useful for the study of membrane proteins in a lipid bilayer environment. Despite its unique advantages, technological hurdles have kept electron crystallography from reaching its full potential. Addressing the issues, recent initiatives developed high-throughput pipelines for crystallization and screening. Adding progress in automating data collection, image analysis and phase extension methods, electron crystallography is poised to raise its profile and may lead the way in exploring the structural biology of macromolecular complexes.     

X-ray diffraction from membrane protein nanocrystals

Membrane proteins constitute >30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is <300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 μm. The results demonstrate that there are membrane protein crystals that contain <100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain <200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells.     

Growth of Large and Highly Ordered 2D Crystals of a K+ Channel,Structural Role of Lipidic Environment

2D crystallography has proven to be an excellent technique to determine the 3D structure of membrane proteins. Compared to 3D crystallography, it has the advantage of visualizing the protein in an environment closer to the native one. However, producing good 2D crystals is still a challenge and little statistical knowledge can be gained from literature. Here, we present a thorough screening of 2D crystallization conditions for a prokaryotic inwardly rectifying potassium channel (>130 different conditions). Key parameters leading to very large and well-organized 2D crystals are discussed. In addition, the problem of formation of multilayers during the growth of 2D crystals is also addressed. An intermediate resolution projection map of KirBac3.1 at 6 Å is presented, which sheds (to our knowledge) new light on the structure of this channel in a lipid environment.     

融合标签技术在膜蛋白结构研究中的应用

膜蛋白高级结构的研究包括不同的层次,即膜蛋白拓扑学结构的研究、利用核磁共振技术和蛋白质晶体衍射技术对三维结构的研究,以及膜蛋白复合体的研究。在研究过程中,如果能够基于膜蛋白的拓扑学结构预测,选择合适的蛋白质或多肽融合标签,利用基因融合技术在基因水平上对膜蛋白进行改造,可以产生含有融合标签的重组膜蛋自,不仅具有原有膜蛋白的功能活性,还具有融合标签所特有的生理生化特性,将会极大地促进膜蛋白结构和功能的研究。我们就目前膜蛋白结构研究中所涉及的融合标签技术及其应用策略和所取得的进展做一简述。     

Stability of membrane proteins: relevance for the selection of appropriate methods for high-resolution structure determinations

High stability is a prominent characteristic of integral membrane proteins of known atomic structure. But rather than being an intrinsic property, it may be due to a selection exerted by biochemical procedures prior to structure determination, since solubilization results in the transient exposure of membrane proteins to solution conditions. This may cause structural perturbations that interfere with 3D crystallization and hence with X-ray analysis. This problem also affects the preparation of samples for electron crystallography and NMR studies and may account for the fact that high-resolution structures of representatives of whole groups, such as transport proteins and signal transducers, have not been elucidated so far by any method. A knowledge of the proportion of labile proteins among membrane proteins, and of the kinetics of their denaturation, is therefore necessary. Establishing stability profiles, developing methods to maintain lateral pressure, or preventing contact with water (or both) should prove significant in establishing the structures of conformationally flexible proteins.     

In vitro selection and characterization of DARPins and Fab fragments for the co-crystallization of membrane proteins: The Na(+)-citrate symporter CitS as an example

The determination of 3D structures of membrane proteins is still extremely difficult. The co-crystallization with specific binding proteins may be an important aid in this process, as these proteins provide rigid, hydrophilic surfaces for stable protein-protein contacts. Also, the conformational homogeneity of the membrane protein may be increased to obtain crystals suitable for high resolution structures. Here, we describe the efficient generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) as specific binding molecules for membrane proteins. We used both phage display and ribosome display to select DARPins in vitro that are specific for the detergent-solubilized Na(+)-citrate symporter CitS of Klebsiella pneumoniae. Compared to classical hybridoma technology, the in vitro selection systems allow a much better control of the structural integrity of the target protein and allow the use of other protein classes in addition to recombinant antibodies. We also compared the selected DARPins to a Fab fragment previously selected by phage display and demonstrate that different epitopes are recognized, unique to each class of binding molecules. Therefore, the use of several classes of binding molecules will make suitable crystal formation and the determination of their 3D structure more likely.     

Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins.

A considerable interest exists currently in designing innovative strategies to produce two-dimensional crystals of membrane proteins that are amenable to structural analysis by electron crystallography. We have developed a protocol for crystallizing membrane protein that is derived from the classical lipid-layer two-dimensional crystallization at the air/water interface used so far for soluble proteins. Lipid derivatized with a Ni(2+)-chelating head group provided a general approach to crystallizing histidine-tagged transmembrane proteins. The processes of protein binding and two-dimensional crystallization were analyzed by electron microscopy, using two prototypic membrane proteins: FhuA, a high-affinity receptor from the outer membrane of Escherichia coli, and the F(0)F(1)-ATP synthase from thermophilic Bacillus PS3. Conditions were found to avoid solubilization of the lipid layer by the detergent present with the purified membrane proteins and thus to allow binding of micellar proteins to the functionalized lipid head groups. After detergent removal using polystyrene beads, membrane sheets of several hundreds of square micrometers were reconstituted at the interface. High protein density in these membrane sheets allowed further formation of planar two-dimensional crystals. We believe that this strategy represents a new promising alternative to conventional dialysis methods for membrane protein 2D crystallization, with the additional advantage of necessitating little purified protein.     

Revival of electron crystallography

Since the structure determination of bacteriorhodopsin in 1990, much progress has been made in the further development and use of electron crystallography. In this review, we provide a concise overview of the new developments in electron crystallography concerning 2D crystallization, data collection and data processing. Based on electron crystallographic work on bacteriorhodopsin, the acetylcholine receptor and aquaporins, we highlight the unique advantages and future perspectives of electron crystallography for the structural study of membrane proteins. These advantages include the visualization of membrane proteins in their native environment without detergent-induced artifacts, the trapping of different states in a reaction pathway by time-resolved experiments, the study of non-specific protein-lipid interactions and the characterization of the charge state of individual residues in membrane proteins.