Membrane Proteins - Engineering, Purification and Crystallization

1. Auflage 2015
ISBN: 978-0-12-802193-4
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 557, 644 Seiten

Reihe: Methods in Enzymology

Membrane Proteins - Engineering, Purification and Crystallization, a volume of Methods In Enzymology, encompasses chapters from the leading experts in the area of membrane protein biology. The chapters provide a brief overview of the topics covered and also outline step-by-step protocol for the interested audience. Illustrations and case example images are included wherever appropriate to help the readers understand the schematics and general experimental outlines. - Volume of Methods In Enzymology - Contains a collection of a diverse array of topics in the area of membrane protein biology ranging from recombinant expression, isolation, functional characterization, biophysical studies and crystallization

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Chapter One Multicolor Fluorescence-Based Screening Toward Structural Analysis of Multiprotein Membrane Complexes
Simon Trowitzsch*; Robert Tampé*,†,1    * Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Frankfurt/Main, Germany
† Cluster of Excellence—Macromolecular Complexes, Goethe-University Frankfurt, Frankfurt/Main, Germany
1 Corresponding author: email address: [e-mail] Abstract
Structures of membrane protein complexes provide a wealth of information on their biological function, the interplay among their subunits, and on ligand binding. Structural genomics of multiprotein membrane complexes seek to deliver structural information ideally of most of these complexes. Models of abundant native membrane protein complexes have been proposed from X-ray crystallography or single-particle cryo-EM. However, most of the remaining membrane protein complexes persist in very low copy numbers per cell and cannot be isolated from their native source without tremendous efforts. Therefore, heterologous expression systems are continually being developed to overproduce membrane protein complexes in various host cells of bacterial or eukaryotic origin. Still, only a small fraction of membrane proteins is suitable for structure determination due to poor expression levels, misfolding and aggregation, complex heterogeneity, imbalanced stoichiometry, and difficulties in solubilization as well as stabilization of the complexes. Powerful tools are therefore necessary to identify the correct expression host and to validate extraction and purification strategies for a given membrane protein complex at the earliest time point. Here, we discuss a fluorescence-based screening approach particularly tailored for the handy and sensitive analysis of the production and purification process for multiprotein membrane complexes. Multicolor fluorescence-detection size-exclusion chromatography provides a powerful readout system and allows quantitative monitoring of the production of critical single subunits of membrane protein complexes. The approach facilitates the tracking of improvements during sample optimization for monodispersity, balanced stoichiometry, and stability of multisubunit membrane protein complexes. Keywords Multisubunit membrane protein complexes Structural biology Multicolor fluorescence-detection size-exclusion chromatography Heterologous expression systems Fluorescence-based screening Peptide-loading complex Transporter associated with antigen processing ABC transporters 1 Introduction
Membrane proteins and membrane protein complexes perform a wide range of essential biological functions including metabolism, signal transduction, energy conversion and utilization. They represent with more than 50% the largest class of potential novel protein drug targets (Arinaminpathy, Khurana, Engelman, & Gerstein, 2009; Russell & Eggleston, 2000; Yildirim, Goh, Cusick, Barabasi, & Vidal, 2007). Although membrane proteins make up nearly 30% of all proteins in the cell, they only constitute ~ 2% of all structures deposited in the Protein Data Bank (PDB, www.rcsb.org). Only a few membrane proteins are abundant in cells, such as mammalian and bacterial rhodopsins, aquaporins, respiratory complexes, F/V/P-type ATPases, photosynthetic complexes, reaction centers, and light harvesting proteins (Bill et al., 2011). The first published structure of an integral membrane protein complex was the photosynthetic reaction center of Rhodopseudomonas viridis extracted from native sources in 1985 (Deisenhofer, Epp, Miki, Huber, & Michel, 1985). And still nowadays, significant numbers of structures solved from endogenous material are deposited in the PDB each year (Buschmann et al., 2010; Efremov, Baradaran, & Sazanov, 2010; Lee, Stewart, Donohoe, Bernal, & Stock, 2010; Mesa, Deniaud, Montoya, & Schaffitzel, 2013; von der Hocht et al., 2011). However, low abundance of the vast majority of membrane proteins renders overexpression essential for large-scale production for structural studies, because milligram quantities of pure, monodisperse, and stoichiometric sample are typically needed. The production of high-quality samples is not trivial though. It took more than a decade after the structural characterization of the photosynthetic reaction center until the first crystal structure of a membrane protein heterologously expressed in Escherichia coli was solved (Doyle et al., 1998). Since then, rigorous efforts to prepare homogeneous and active recombinant samples for structural investigation have lead to the determination of around 500 unique structures of membrane proteins and membrane protein complexes at atomic or near atomic resolution (Fig. 1; http://blanco.biomol.uci.edu/mpstruc/). Figure 1 Classification of released unique membrane protein structures in the PDB based on the source organism for sample preparation (either recombinant or from a native source). (A) The histogram shows the distribution of unique membrane protein structures from all domains of life as a plot against the expression host, from which the sample was derived. Inset: a pie chart showing the distribution of the genetic origin of the membrane protein structures. (B) The histogram summarizes the distribution of unique eukaryotic, recombinant membrane protein structures as a plot against the expression host. Inset: a pie chart showing the distribution of the number of subunits for each unique eukaryotic, recombinant membrane protein structure. A number of heterologous expression systems have been used and optimized for the production of membrane proteins, such as bacterial and yeast expression systems, baculovirus-based insect cell systems, and cultured mammalian cells (Andrell & Tate, 2013; Bonander & Bill, 2012; Condreay & Kost, 2007; Frelet-Barrand, Boutigny, Kunji, & Rolland, 2010; Junge et al., 2008; Sahdev, Khattar, & Saini, 2008). Cell-free protein production is also being used and constantly developed for membrane protein synthesis for structural analysis (Harbers, 2014). Engineered baculoviruses are promising versatile vectors, not only for protein production in insect cells but also for transducing numerous types of mammalian cells, thus facilitating the search for the best expression host (Kost, Condreay, & Jarvis, 2005; Liu, Chen, & Chao, 2010; Sung et al., 2014). Not surprisingly, eukaryotic expression systems are becoming more and more important as expression hosts for the production of membrane proteins of eukaryotic origin (Fig. 1). Although approximately 38% of heterologously expressed eukaryotic membrane protein structures were determined using samples produced in E. coli, the insect cell expression system including cell lines from Spodoptera frugiperda and Trichoplusia ni is turning out to be a very attractive approach (He, Wang, & Yan, 2014). Yeast and mammalian systems rank third and fourth accounting for ~ 20% and 4% of the unique structures deposited, respectively. Identifying the best expression host for a given eukaryotic membrane protein or membrane protein complex is still an empirical process and usually takes a significant amount of time and resources (Bernaudat et al., 2011). In order to overcome a critical bottleneck in structure determination of membrane proteins, high-throughput approaches have been developed and implemented but only show success rates of ~ 0.3% (Mancia & Love, 2010). The main problem is that conditions need to be optimized toward stability and homogeneity for each specimen in order to obtain a conformationally rigid sample, which can eventually intercalate into a regular crystal lattice for structure determination by X-ray crystallography. Recent technological advances in direct electron detection cameras and innovative image processing algorithms enabled structure determination by single-particle cryo-electron microscopy (cryo-EM) at near atomic resolution (Allegretti, Mills, McMullan, Kühlbrandt, & Vonck, 2014; Amunts et al., 2014; Bai, Fernandez, McMullan, & Scheres, 2013; Kim et al., 2014; Li et al., 2013). Of note is the first structure of a heterologously expressed homotetrameric ion channel, TRPV1, extracted from transiently transfected HEK293S GnTI- cells (Liao, Cao, Julius, & Cheng, 2013). Another milestone for structural analysis of heterologously produced multiprotein membrane complexes by single-particle cryo-EM is the 3D reconstruction of the heterotetrameric ?-secretase at 4.5 Å resolution and the heterodimeric ABC transporter TmrAB (Kim et al., 2014; Lu et al., 2014). One has yet to bear in mind that sample preparation and structure determination of these complexes took several years and that rigorous efforts were made to obtain homogeneous, active sample for structural investigation. Taking into account all the difficulties during production, purification, and stabilization already encountered for a single recombinant membrane protein, it is not surprising that only a few structures of hetero-oligomeric eukaryotic...