High-throughput identification of purification conditions leads to preliminary crystallization conditions for three inner membrane proteins

An important factor in the crystallization, and subsequent structural determination, of integral membrane proteins is the ability to produce a stable and monodisperse solution of the protein. Obtaining the correct purification detergent to achieve this can be laborious and is often serendipitous. In this study, high-throughput methods are used to analyze the suitability of eight different detergents on the stability of 12 inner transmembrane proteins from Escherichia coli. The best results obtained from the small-scale experiments were scaled up, the aggregation state of the proteins assessed, and all monodisperse protein solutions entered into crystallization trials. This resulted in preliminary crystallization hits for three inner membrane proteins: XylH, PgpB and YjdL and this study reports the methods, purification procedures and crystallization conditions used to achieve this.     

Strategies for crystallizing membrane proteins

Crystallizing membrane proteins remains a challenging endeavor despite the increasing number of membrane protein structures solved by X-ray crystallography. The critical factors in determining the success of the crystallization experiments are the purification and preparation of membrane protein samples. Moreover, there is the added complication that the crystallization conditions must be optimized for use in the presence of detergents although the methods used to crystallize most membrane proteins are, in essence, straightforward applications of standard methodologies for soluble protein crystallization. The roles that detergents play in the stability and aggregation of membrane proteins as well as the colloidal properties of the protein-detergent complexes need to be appreciated and controlledbefore and during the crystallization trials. All X-ray quality crystals of membrane proteins were grown from preparations of detergent-solubilized protein, where the heterogeneous natural lipids from the membrane have been replaced by ahomogeneous detergent environment. It is the preparation of such monodisperse, isotropic solutions of membrane proteins that has allowed the successful application of the standard crystallization methods routinely used on soluble proteins. In this review, the issues of protein purification and sample preparation are addressed as well as the new refinements in crystallization methodologies for membrane proteins. How the physical behavior of the detergent, in the form of micelles or protein-detergent aggregates, affects crystallization and the adaptation of published protocols to new membrane protein systems are also addressed. The general conclusion is that many integral membrane proteins could be crystallized if pure and monodisperse preparations in a suitable detergent system can be prepared.In memory of Glenn D. Garavito.     

Application of Mistic to improving the expression and membrane integration of histidine kinase receptors from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Integral membrane proteins have become the focus of interest of many laboratories and structural genomics consortia, but their study is hampered by bottlenecks in production, solubilization, purification and crystallization. In our laboratory we have addressed the problem of high-level protein expression in the membrane of Escherichia coli by use of Mistic, a novel Bacillus subtilis protein, as a fusion partner. In this study we examine the effect of Mistic on protein expression and membrane integration levels of members of the E. coli histidine kinase receptor family. We find that Mistic fusion invariably increases the overall yield by targeting the cargo proteins more efficiently to the membrane and may even replace the signal sequence. Mistic fusion methods will likely be instrumental for high-level expression of other integral membrane proteins.     

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation

Abstract  Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.     

Rational design of a fusion partner for membrane protein expression in E. coli

We have designed a novel protein fusion partner (P8CBD) to utilize the co‐translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP‐dependence was demonstrated by analyzing the membrane translocation of P8CBD‐PhoA fusion proteins in wt and SRP‐ffh77 mutant cells. We also demonstrate that the P8CBD N‐terminal fusion partner promotes over‐expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over‐expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane‐associated protein.     

Eukaryotic integral membrane protein expression utilizing the <Emphasis Type="Italic">Escherichia coli</Emphasis> glycerol-conducting channel protein (GlpF)

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).     

膜蛋白结晶方法学研究进展

膜蛋白执行着物质运输、能量转换和信号转导等重要生物学功能,其分子的三维结构解析对阐述其功能及开展理性药物设计有着十分重要的意义．目前膜蛋白结构解析以X射线单晶衍射技术为主,该技术需要高质量晶体作为衍射对象．然而由于膜蛋白具有两亲性,难以得到高度有序的三维晶体,进而导致其结构解析十分困难．针对此问题,研究者们发展了一些专门面向膜蛋白的结晶方法,如基于去垢剂的方法,基于脂类的方法等．本文回顾了这些方法,并对未来膜蛋白的结晶研究进行了展望．     

New techniques for membrane protein crystallization tested on photosystem II core complex of Pisum sativum

The crystallization of a given protein is a hard task being even more complicated when the protein shows a hydrophobic behavior. In the case of photosynthetic proteins, the difficulty of the experiments increased due to the high light sensitivity. Aqueous solutions of photosystem II core complex (OEC PSII) of Pisum sativum were screened for crystallization conditions using standard crystallization methods. Crystal improvement was achieved by counter-diffusion technique in single capillaries of 0.2 mm inner diameter with a three-layer configuration. The use of this advanced crystallization technique—for the first time applied to the crystallization of membrane proteins—improves the reproducibility of the experiments allowing the initial crystal characterization, and facilitates the manipulation under light protection.     

膜蛋白晶体的生长及其进展

生物膜中与脂双层结合的蛋白质称为膜蛋白.由于它们具有很大的疏水表面以及既亲水又疏水的两性特点致使其纯化与结晶都十分困难.在膜蛋白晶体生长系统中引入小分子去污剂与小的两性分子获得突破性进展.迄今为止,结晶出来的膜蛋白为数不多.其中只有光合细菌绿色红假单胞菌及球型红假单胞菌的反应中心得到3分辨率的晶体结构与解析.一系列膜蛋白形成二维晶体,可用电子显微镜与像重构技术获得三维结构信息.     

Enrichment of hydrophobic membrane proteins using Triton X-114 and subsequent analysis of their N-glycosylation

BackgroundNumerous proteins depend on correct glycosylation for their proper function and nearly all membrane, as well as secreted, proteins are glycosylated. Glycosylation of membrane proteins plays a crucial role in many processes including the intercellular recognition and intermolecular interactions on the cell surface. The composition of N-glycans attached to membrane proteins has not been sufficiently studied due to the lack of efficient and reproducible analytical methods.MethodsThe aim of this study was to optimise cloud-point extraction (CPE) of membrane proteins with the non-ionic detergent Triton X-114 and analyse their N-glycosylation using hydrophilic interaction liquid chromatography (HILIC-UPLC). Purification of isolated proteins from the excess of detergent proved to be the key step. Therefore, several purification procedures were tested to efficiently remove detergent, while retaining maximum protein recoveries.ResultsCPE showed to be an efficient method to simultaneously extract membrane and soluble proteins, which subsequently resulted in different N-glycan profiles of the aforementioned protein groups. The resulting protocol showed satisfactory reproducibility and potential for N-glycan analysis of both membrane and intracellular (soluble) proteins from different kinds of biological material.ConclusionsThis method can be used as a new analytical tool for reliable detection and quantification of oligomannose and complex type N-glycans attached to membrane proteins, thus serving to distinguish between differences in cell types and states.General significanceThe simple method was successfully optimised to generate reliable HILIC-UPLC profiles of N-glycans released from membrane proteins. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

副溶血性弧菌脂蛋白定位系统转运蛋白结构与功能的生物信息学分析

【目的】副溶血性弧菌是一种重要的人畜共患病原菌,脂蛋白定位系统(Localization of lipoprotein system,Lol)负责该菌脂蛋白的转运与定位,与其致病力及耐药性密切相关,对Lol系统转运蛋白进行系统的生物信息学分析,有助于推动副溶血性弧菌致病与耐药机理的进一步研究。【方法】本文通过生物信息学分析技术,结合ExPASy在线工具、SignalP 4.0 Server、TMHMM-2.0、STRING、SWISS-MODEL等软件,分析了副溶血性弧菌Lol系统转运蛋白LolA-E及LolCD_2E的基本性质、蛋白互作关系及三级结构。【结果】LolA和LolB为酸性亲水蛋白,含信号肽位点,无跨膜区域。LolC和LolE为碱性疏水膜蛋白,LolCD_2E为中性疏水膜蛋白,LolC-E及LolCD_2E均无显著的信号肽位点。蛋白相互作用网络显示,LolA–E五个蛋白的编码基因均共表达,负责脂蛋白的合成与转运,并与BamA、Pal、MacB、CmeC等外膜蛋白具有密切的互作关系。三级结构同源建模发现,副溶血性弧菌与大肠杆菌拥有相似的LolA和LolB结构,LolC-E含有MacB蛋白的同源结构,赋予了该系统消耗ATP运输脂蛋白的重要功能。此外,本研究还首次发现了副溶血性弧菌LolC和LolE中存在一段保守的Hook结构,是LolCD_2E复合物与LolA结合并转运脂蛋白的关键区域。【结论】本研究为副溶血性弧菌Lol系统转运蛋白的表达纯化、结构与功能的研究提供了重要的数据基础,为后续抗菌药物的研发提供了新型作用靶点。     

Engineering the lac permease for purification and crystallization

The lactose permease is being used as a model system for the rational redesign of a membrane protein with the goal of increasing the likelihood of crystallization. Various modifications to the protein have been added for the purposes of purification, stability, and potential for crystallization. The addition of six consecutive histidines at the C-terminus of the protein allows for the rapid purification by nickel-chelate chromatography, and the insertion of an entire protein domain into one of the inner cytoplasmic loops of the permease gives the resulting protein a larger hydrophilic surface area. The increase in polar surface area makes the fusion protein easier to handle and more likely to crystallize. In particular, the introduction of cytochromeb562 ofE. coli into the central hydrophilic domain of the lac permease results in a fusion protein with the transport activity of the permease and the visible absorbance spectrum of the cytochrome. The red permease is very easy to monitor through the steps of expression, purification, concentration, and crystallization.     

Crystallization of the membrane protein hVDAC1 produced in cell-free system

Structural studies of membrane proteins are in constant evolution with the development of new improvements for their expression, purification, stabilization and crystallization. However, none of these methods still provides a universal approach to solve the structure of membrane proteins. Here we describe the crystallization of the human voltage-dependent anion channel-1 produced by a bacterial cell-free expression system. While VDAC structures have been recently solved, we propose an alternative strategy for producing the recombinant protein, which can be applied to other membrane proteins reluctant to expression, purification and crystallization by classical approaches. Despite a lot of efforts to crystallize a cell-free expressed membrane protein, this study is to our knowledge one of the first reports of a successful crystallization. Focusing on expression in a soluble and functional state, in a detergent environment, is the key to get crystals. Although the diffraction of VDAC crystals is limited, the simplicity and the rapidity to set-up and optimize this technology are drastic advantages in comparison to other methods.     

Cloning,expression and purification of orthologous membrane proteins: a general protocol for preparation of the histidine sensor kinase ETR1 from different species

Abstract

Orthologous proteins do not necessarily share the same function in all species and those sharing the same function might employ a modified catalytic mechanism. Thus, comparative analysis of homologous or orthologous proteins from different organisms can provide detailed information on the function and the mechanism of an entire protein family. The sensor kinase ETR1 from Arabidopsis thaliana has been well characterized by genetic, physiological and biochemical studies. However, as further model plants are coming into focus for plant hormone research, a general protocol for isolation and purification of orthologous ETR1 proteins seems instrumental for a detailed molecular analysis of this protein family. In this study, we describe the native purification of recombinant ETR1 from Arabidopsis thaliana by mild solubilization with the zwitter-ionic detergent Fos-Choline-14 and single-step purification by immobilized metal ion affinity chromatography. The same protocol was successfully applied for the purification of the orthologous proteins from the moss Physcomitrella patens subsp. patens and the tomato Lycopersicon esculentum. The successful transfer of the purification protocol to proteins of the same family which share sequence identity of 63–80% only suggests that this protocol presents a general purification strategy which is likely to apply also to the purification of other members of the sensor histidine kinase family.     

Rapid Isolation of Single-chain Antibodies for Structural Genomics

High throughput approaches to structural genomics requires expression, purification, and crystallization of proteins derived from predicted open reading frames cloned into a host organism, typically E. coli. Early results from this approach suggest that the success rate of obtaining well diffracting crystals from eukaryotic proteins is disappointingly low. A proven method of improving the odds of crystallization is formation of a complex with a conformation-stabilizing partner of known structure that is easily crystallized. Such complexes are also able to engage in different crystal contacts than the original protein by itself. Fab fragments derived from monoclonal antibodies have been successfully used for this purpose for a variety of proteins, however conventional methods for the isolation of monoclonal antibodies from hybridomas are time consuming and expensive. We are exploring the use of phage display to generate recombinant antibodies to target proteins that can be used to obtain co-complexes to facilitate crystallization and structural determination. We are using a large, human single-chain Fv (scFv) library to select for antibodies that bind to a panel of Leishmania major target proteins. Thirteen out of 16 target proteins yielded good binders after three rounds of enrichment. A total of 55 distinct scFvs were identified, with five targets each yielding at least five different scFvs. Individual clones were analyzed for binding specificity and soluble scFv can be readily produced and purified via the appended His₆ epitope tag. Using immunoaffinity chromatography, eight scFv target protein pairs were identified that exhibit stable complex formation and are suitable for co-crystallization trials.     

Combinatorial Method for Overexpression of Membrane Proteins in Escherichia coli

Membrane proteins constitute 20–30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.     

Structures of the OmpF porin crystallized in the presence of foscholine‐12

The endogenous Escherichia coli porin OmpF was crystallized as an accidental by‐product of our efforts to express, purify, and crystallize the E. coli integral membrane protein KdpD in the presence of foscholine‐12 (FC12). FC12 is widely used in membrane protein studies, but no crystal structure of a protein that was both purified and crystallized with this detergent has been reported in the Protein Data Bank. Crystallization screening for KdpD yielded two different crystals of contaminating protein OmpF. Here, we report two OmpF structures, the first membrane protein crystal structures for which extraction, purification, and crystallization were done exclusively with FC12. The first structure was refined in space group P21 with cell parameters a = 136.7 Å, b = 210.5 Å, c = 137 Å, and β = 100.5°, and the resolution of 3.8 Å. The second structure was solved at the resolution of 4.4 Å and was refined in the P321 space group, with unit cell parameters a = 215.5 Å, b = 215.5 Å, c = 137.5 Å, and γ = 120°. Both crystal forms show novel crystal packing, in which the building block is a tetrahedral arrangement of four trimers. Additionally, we discuss the use of FC12 for membrane protein crystallization and structure determination, as well as the problem of the OmpF contamination for membrane proteins overexpressed in E. coli.     

A protocol for high throughput methods for the expression and purification of inner membrane proteins

The production of diffraction quality crystals for the structural determination of inner membrane proteins relies on obtaining large amounts of stable protein. Achieving this, by finding the correct parameters to successfully express and purify these proteins is often time-consuming and frustrating. The methods described here examine the most important parameters, in both expression and purification, quickly and simply. They take into account methods previously used in successful structural determinations of inner membrane proteins and collect and analyse data for use in further experiments and to investigate overall trends. These methods make use of histidine-tagged membrane proteins with a green fluorescent protein fusion but could be adapted easily for other proteins.     

细胞因子人脂联素全序列蛋白的真核表达及晶体生长

目的:找寻适用于脂联素全序列蛋白的结晶条件,为解析其空间结构奠定基础,从而研究脂联素聚合体的内在构成模式,为开发高活性脂联素类衍生细胞因子提供参考。方法:首先构建脂联素全序列蛋白的真核表达载体,对其进行诱导表达,然后通过经亲和层析和凝胶过滤分离纯化后,得到高纯度的脂联素全序列蛋白,最后尝试使用坐滴法和悬滴法以及多种温度环境和结晶液条件,从而找寻适于脂联素全序列蛋白质的结晶条件。结果:通过纯化后的脂联素蛋白纯度可以达到91.3%,在溶液中的粒径分布于2 nm到4 nm。在线性变温条件下(24 h内,由277 K线性升温至313 K,再线性降温至277 K),通过悬滴法于48 h可获得脂联素全序列蛋白的针状晶体。结论:本研究选择真核载体,以亲和层析和凝胶过滤为分离纯化手段,得到了纯度高,粒径均一的脂联素全序列蛋白。随后通过尝试多种结晶方法、条件和环境,初步确定获得脂联素全序列蛋白晶体的条件,为后续获得高质量单晶提供了参考。     

Camelid nanobodies raised against an integral membrane enzyme,nitric oxide reductase

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N₂O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies™). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.