Overexpression of integral membrane proteins for structural studies

Grisshammer, Reinhard; Tate, Christopher G.

doi:10.1017/s0033583500003504

Cited by 369 publications

(275 citation statements)

References 424 publications

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“…In addition, this gene was previously reported to be the human ortholog of the porcine receptor for endogenous retrovirus A, which mediates the infection of the cells with porcine endogenous retrovirus (6). Because its amino acid sequence is similar to that of a G protein-coupled receptor family member and a multimembrane spanning protein, it is anticipated that it will be difficult to produce a functional antibody against hRFT1, as previously reported (9,22,39). In the present study, EGFP-tagged hRFT1 was expressed in the plasma membrane (Fig.…”

Section: Discussionmentioning

confidence: 88%

Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

Yonezawa¹,

Masuda²,

Katsura³

et al. 2008

American Journal of Physiology-Cell Physiology

127

101

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Section: Discussionmentioning

confidence: 88%

Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

Yonezawa¹,

Masuda²,

Katsura³

et al. 2008

American Journal of Physiology-Cell Physiology

127

101

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“…This situation fundamentally reflects the limitations of current systems for the overexpression of membrane proteins of relatively low abundance (17). As a consequence, most membrane proteins of known structure are naturally present in relatively high abundance so that overexpression methods are not essential.…”

Section: Structural Analysis Of Kcsa and Msclmentioning

confidence: 99%

Crystallographic Analyses of Ion Channels: Lessons and Challenges

Rees

Chang²,

Spencer

2000

Journal of Biological Chemistry

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Membrane proteins fascinate at many levels, from their central functional roles in transport, energy transduction, and signal transduction processes to structural questions concerning how they fold and operate in the exotic environments of the membrane bilayer and the water-bilayer interface and to methodological issues associated with studying membrane proteins either in situ or extracted from the membrane. This interplay is beautifully exemplified by ion channels, a collection of integral membrane proteins that mediate the transmembrane passage of ions down their electrochemical potential gradient (for general reviews, see Refs. 1 and 2). Ion channels are key elements of signaling and sensing pathways, including nerve cell conduction, hormone response, and mechanosensation. The characteristic properties of ion channels reflect their conductance, ion selectivity, and gating. Ion channels are often specific for a particular type of ion (such as potassium or chloride) or a class of ions (such as anions) and are typically regulated by conformational switching of the protein structure between "open" and "closed" states. This conformational switching may be gated in response to changes in membrane potential, ligand binding, or application of mechanical forces. Detailed functional characterizations of channels and their gating mechanisms have been achieved, reflecting exquisite methodological advances such as patch clamp methods that can monitor the activities of individual channels (3). Until recently, corresponding information about the three-dimensional structures of channels was not available, reflecting difficulties in obtaining sufficient quantities of membrane proteins for crystallization trials. Happily, this situation has started to change with the structure determinations of the Streptomyces lividans K ϩ channel (KcsA (4)) and the Mycobacterium tuberculosis mechanosensitive channel (MscL (5)).A variety of reviews (6 -12) have appeared recently that discuss functional implications of these channel structures. This review discusses these developments from a complementary perspective, by considering the implications of these structures from within the larger framework of membrane protein structure and function. Because of space restrictions, this review necessarily emphasizes membrane proteins that are composed primarily of ␣-helical bundles, such as KcsA and MscL, rather than ␤-barrel proteins, such as porins, typically found in bacterial outer membranes. What Are KcsA and MscL? A Brief IntroductionKcsA and MscL are prokaryotic channels that fold as homooligomers (tetramers and pentamers, respectively) of relatively small subunits that contain two transmembrane-spanning helices. KcsA is a potassium-selective channel, consisting of a 160-amino acid subunit, that was identified in S. lividans by Schrempf et al. (13). KcsA shares the signature sequences with eukaryotic K ϩ channels that are responsible for ion selectivity and pore formation. However, this prokaryotic channel lacks the regions of eukaryotic chan...

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“…Bacterial membrane proteins have been easier to overexpress using standard techniques in Escherichia coli than eukaryotic membrane proteins (2,3), and the bacterial proteins are often far more stable in detergent, detergent stability being an essential prerequisite to purification and crystallization. However, of the 125 unique membrane protein structures that have been solved to date, there are only eight structures of mammalian integral membrane proteins; five of these membrane proteins were purified from natural sources and are stable in detergent solutions.…”

mentioning

confidence: 99%

Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form

Serrano-Vega

Magnani

Shibata

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

404

416

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There are Ϸ350 non-odorant G protein-coupled receptors (GPCRs) encoded by the human genome, many of which are predicted to be potential therapeutic targets, but there are only two structures available to represent the whole of the family. We hypothesized that improving the detergent stability of these receptors and simultaneously locking them into one preferred conformation will greatly improve the chances of crystallization. We developed a generic strategy for the isolation of detergent-solubilized thermostable mutants of a GPCR, the ␤1-adrenergic receptor. The most stable mutant receptor, ␤AR-m23, contained six point mutations that led to an apparent Tm 21°C higher than the native protein, and, in the presence of bound antagonist, ␤AR-m23 was as stable as bovine rhodopsin. In addition, ␤AR-m23 was significantly more stable in a wide range of detergents ideal for crystallization and was preferentially in an antagonist conformation in the absence of ligand.G protein-coupled receptor ͉ membrane protein ͉ stabilization O ver the past 20 years the rate of determination of membrane protein structures has gradually increased, but most success has been in crystallizing membrane proteins from bacteria rather than from eukaryotes (1). Bacterial membrane proteins have been easier to overexpress using standard techniques in Escherichia coli than eukaryotic membrane proteins (2, 3), and the bacterial proteins are often far more stable in detergent, detergent stability being an essential prerequisite to purification and crystallization. However, of the 125 unique membrane protein structures that have been solved to date, there are only eight structures of mammalian integral membrane proteins; five of these membrane proteins were purified from natural sources and are stable in detergent solutions. Apart from the difficulties in overexpressing eukaryotic membrane proteins, they often have poor stability in detergent solutions, which severely restricts the range of crystallization conditions that can be explored without their immediate denaturation or precipitation. Ideally, membrane proteins should be stable for many days in any given detergent solution, but the detergents that are best suited to growing diffraction-quality crystals tend to be the most destabilizing detergents, i.e., those with short aliphatic chains and small or charged head groups. It is also the structures of human membrane proteins that we would like to solve, because these are required to help the development of therapeutic agents by the pharmaceutical industry; often there are substantial differences in the pharmacology of receptors, channels, and transporters from different mammals, whereas yeast and bacterial genomes may not include any homologous genes. There is thus an overwhelming need to develop a generic strategy that will allow the production of detergent-stable eukaryotic integral membrane proteins for crystallization and structure determination.Membrane proteins have evolved to be sufficiently stable in the membrane to ensure cell viability, b...

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Overexpression of integral membrane proteins for structural studies

Cited by 369 publications

References 424 publications

Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

Crystallographic Analyses of Ion Channels: Lessons and Challenges

Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form

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