International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 4.2, p. 94   | 1 | 2 |

Section 4.2.1. Introduction

H. Michela*

aMax-Planck-Institut für Biophysik, Heinrich-Hoffmann-Strasse 7, D-60528 Frankfurt/Main, Germany
Correspondence e-mail: michel@mpibp-frankfurt.mpg.de

4.2.1. Introduction

| top | pdf |

At the time of writing, the Protein Data Bank contains more than 8000 entries of protein structures. These belong to roughly 1200 sequence-unrelated protein families, which can be classified into ∼350 different folds (Gerstein, 1998[link]). However, all known membrane-protein structures belong to one of a dozen membrane-protein families. Membrane proteins for which the structures have been determined are bacterial photosynthetic reaction centres, porins and other β-strand barrel-forming proteins from the outer membrane of Gram-negative bacteria, bacterial light-harvesting complexes, bacterial and mitochondrial cytochrome c oxidase, the cytochrome [bc_{1}] complex from mammalian mitochondria, two cyclooxygenases, squalene cyclase, and two bacterial channel proteins. These structures have been determined by X-ray crystallography. In addition, the structures of two membrane proteins, namely that of bacteriorhodopsin and that of the plant light-harvesting complex II, have been solved by electron crystallography (see Chapter 19.6[link] ). Table 4.2.1.1[link] provides a list of the membrane proteins with known structures. It also contains the key references for the structure descriptions and the crystallization conditions.

Table 4.2.1.1| top | pdf |
Compilation of membrane proteins with known structures, including crystallization conditions and key references for the structure determinations

This table is continuously updated and can be inspected at http://www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html . The membrane proteins listed are divided into polytopic membrane proteins from inner membranes of bacteria and mitochondria (a), membrane proteins from the outer membrane of Gram-negative bacteria (b) and monotopic membrane proteins [(c); these are proteins that are only inserted into the membrane, but do not span it]. Within parts (a), (b) and (c) the membrane proteins are listed in chronological order of structure determination.

(a) Polytopic membrane proteins from inner membranes of bacteria and mitochondria.

Membrane proteinCrystallization conditions (detergent/additive/precipitating agent)Key references (and pdb reference code, if available)
Photosynthetic reaction centre    
 from Rhodopseudomonas viridis N,N-Dimethyldodecylamine-N-oxide/heptane-1,2,3-triol/ammonium sulfate [1], [2] (1PRC), [3], [4] (2PRC, 3PRC, 4PRC, 5PRC, 6PRC, 7PRC)
 from Rhodobacter sphaeroides N,N-Dimethyldodecylamine-N-oxide/heptane-1,2,3-triol/polyethylene glycol 4000 [5] (4RCR)
Octyl-β-D-glucopyranoside/polyethylene glycol 4000 [6] (2RCR)
N,N-Dimethyldodecylamine-N-oxide/heptane-1,2,3-triol, dioxane/potassium phosphate [7] (1PCR)
Octyl-β-D-glucopyranoside/benzamidine, heptane-1,2,3-triol/polyethylene glycol 4000 [8] (1AIG, 1AIJ)
Bacteriorhodopsin    
 from Halobacterium salinarium (Electron crystallography using naturally occuring two-dimensional crystals) [9] (1BRD), [10] (2BRD), [11] (1AT9)
(Type I crystal grown in lipidic cubic phases) [12] (1AP9), [13] (1BRX)
Octyl-β-D-glucopyranoside/benzamidine/sodium phosphate (epitaxic growth on benzamidine crystals) [14] (1BRR)
Light-harvesting complex II    
 from pea chloroplasts (Electron crystallography of two-dimensional crystals prepared from Triton X100 solubilized material) [15]
Light-harvesting complex 2    
 from Rhodopseudomonas acidophila Octyl-β-D-glucopyranoside/benzamidine/phosphate [16] (1KZU)
 from Rhodospirillum molischianum N,N-dimethylundecylamine-N-oxide/heptane-1,2,3-triol/ammonium sulfate [17] (1LGH)
Cytochrome c oxidase    
 from Paracoccus denitrificans,    
 four-subunit enzyme complexed with antibody Fv fragment Dodecyl-β-D-maltoside/polyethylene glycol monomethylether 2000 [18]
 two-subunit enzyme complexed with antibody Fv fragment Undecyl-β-D-maltoside/polyethylene glycol monomethylether 2000 [19] (1AR1)
 from bovine heart mitochondria Decyl-β-D-maltoside with some residual cholate/polyethylene glycol 4000 [20], [21] (1OCC), [22] (2OCC, 1OCR)
Cytochrome bc1 complex    
 from bovine heart mitochondria Decanoyl-N-methylglucamide or diheptanoyl phosphatidyl choline/polyethylene glycol 4000 [23] (1QRC), [24]
Octyl-β-D-glucopyranoside/polyethylene gycol 4000 [25]
Pure dodecyl-β-D-maltoside or mixture with methyl-6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside/polyethylene glycol 4000 [26]
 from chicken heart mitochondria Octyl-β-D-glucopyranoside/polyethylene glycol 4000 [25] (1BCC, 3BCC)
Potassium channel    
 from Streptomyces lividans N,N-Dimethyldodecylamine/polyethylene glycol 400 [27] (1BL8)
Mechanosensitive ion channel    
 from Mycobacterium tuberculosis Dodecyl-β-D-maltoside/triethylene glycol [28]

(b) Membrane proteins from the outer membrane of Gram-negative bacteria and related proteins.

Membrane proteinCrystallization conditions (detergent/additive/precipitating agent)Key references (and pdb reference code, if available)
16-Stranded porins    
 from Rhodobacter capsulatus Octytetraoxyethylene/polyethylene glycol 600 [29] (2POR)
 OmpF and PhoE from Escherichia coli Mixture of n-octyl-2-hydroxyethylsulfoxide and octylpolyoxyethylene; or N,N-dimethyldecylamine-N-oxide/polyethylene glycol 2000 [30] (1OPF, 1PHO), [31]
 from Rhodopseudomonas blastica Octyltetraoxyethylene/heptane-1,2,3-triol/polyethylene glycol 600 [32] (1PRN)
 from Paracoccus denitrificans Octyl-β-D-glucoside/polyethylene glycol 600 [33]
18-Stranded porins    
 maltoporin from Escherichia coli Mixture of decyl-β-D-maltoside and dodecylnonaoxyethylene/polyethylene glycol 2000 [34] (1MAL)
 maltoporin from Salmonella typhimurium Mixture of octyltetraoxyethylene and N,N-dimethylhexylamine-N-oxide/polyethylene glycol 1500 [35] (1MPR, 2MPR)
 sucrose-specific ScrY porin from Salmonella typhimurium Mixture of octyl-β-D-glucopyranoside and N,N-dimethylhexylamine-N-oxide/polyethylene glycol 2000 [36] (1AOS, 1AOT)
α-Haemolysin    
 from Staphylococcus aureus Octyl-β-D-glucopyranoside/ammonium sulfate, polyethylene glycol monomethylether 5000 [37] (7AHL)
Eight-stranded β-barrel membrane anchor    
 OmpA fragment from Escherichia coli Not yet available [38] (1BXW)
22-Stranded receptors    
 FhuA from Escherichia coli N,N-Dimethyldecylamine-N-oxide/inositol/polyethylene glycol monomethylether 2000 [39] (1FCP, 2FCP)
n-Octyl-2-hydroxyethylsulfoxide/polyethylene glycol 2000 [40] (1BY3, 1BY5)
 ferric enterobacterin receptor (FepA) from Escherichia coli N,N-dimethyldodecylamine-N-oxide/heptane-1,2,3-triol/polyethylene glycol 1000 [41] (1FEP)

(c) Proteins inserted into, but not crossing the membrane (`monotopic membrane proteins').

Membrane proteinCrystallization conditions (detergent/additive/precipitating agent)Key references (and pdb reference code, if available)
Prostaglandin H2 synthase 1    
 (cyclooxygenase 1) from sheep Octyl-β-D-glucopyranoside/polyethylene glycol 4000 [42] (1PRH)
Cyclooxygenase 2    
 from mouse Octyl-β-D-glucopyranoside/polyethylene glycol monomethylether 550 [43] (1CX2, 3PGH, 4COX, 5COX, 6COX)
 from man Octylpentaoxyethylene/polyethylene glycol 4000 [44]
Squalene cyclase    
 from Alicyclobacillus acidocaldarius Octyltetraoxyethylene/sodium citrate [45] (1SQC)

References: [1] Diesenhofer et al. (1985[link]); [2] Diesenhofer et al. (1995[link]); [3] Lancaster & Michel (1997[link]); [4] Lancaster & Michel (1999[link]); [5] Allen et al. (1987[link]); [6] Chang et al. (1991[link]); [7] Ermler et al. (1994[link]); [8] Stowell et al. (1997[link]); [9] Henderson et al. (1990[link]); [10] Grigorieff et al. (1996[link]); [11] Kimura et al. (1997[link]); [12] Pebay-Peyroula et al. (1997[link]); [13] Luecke et al. (1998[link]); [14] Essen et al. (1998[link]); [15] Kühlbrandt et al. (1994[link]); [16] McDermott et al. (1995[link]); [17] Koepke et al. (1996[link]); [18] Iwata et al. (1995[link]); [19] Ostermeier et al. (1997[link]); [20] Tsukihara et al. (1995[link]); [21] Tsukihara et al. (1996[link]); [22] Yoshikawa et al. (1998[link]); [23] Xia et al. (1997[link]); [24] Kim et al. (1998[link]); [25] Zhang et al. (1998[link]); [26] Iwata et al. (1998[link]); [27] Doyle et al. (1998[link]); [28] Chang et al. (1998[link]); [29] Weiss et al. (1991[link]); [30] Cowan et al. (1992[link]); [31] Cowan et al. (1995[link]); [32] Kreusch et al. (1994[link]); [33] Hirsch et al. (1997[link]); [34] Schirmer et al. (1995[link]); [35] Meyer et al. (1997[link]); [36] Forst et al. (1998[link]); [37] Song et al. (1996[link]); [38] Pautsch & Schulz (1998[link]); [39] Ferguson et al. (1998[link]); [40] Locher et al. (1998[link]); [41] Buchanan et al. (1999[link]); [42] Picot et al. (1994[link]); [43] Kurumbail et al. (1996[link]); [44] Luong et al. (1996[link]); [45] Wendt et al. (1997[link]).

It is known from genome sequencing projects that 20–35% of all proteins contain at least one transmembrane segment (Gerstein, 1998[link]), as deduced from the occurrence of stretches of hydrophobic amino acids that are long enough to span the membrane in a helical manner. These numbers may be an underestimate, because β-strand-rich membrane proteins like the porins, or membrane proteins which are only inserted into the membrane, but do not span it (`monotopic membrane proteins'), like cyclooxygenases (prostaglandin-H synthases), cannot be recognized as being membrane proteins by inspecting their amino-acid sequences.

Why do we know so few membrane-protein structures? The first reason is the lack of sufficient amounts of biochemically well characterized, homogeneous and stable membrane-protein preparations. This is especially true for eukaryotic receptors and transporters (we do not know the structures of any of these proteins). For these, a major problem is the lack of efficient expression systems for heterologous membrane-protein production. It is therefore not surprising that most membrane proteins with known structure are either involved in photosynthesis or bioenergetics (they are relatively abundant), or originate from bacterial outer membranes (they are exceptionally stable and can be overproduced). Second, membrane proteins are integrated into membranes. They have two polar surface regions on opposite sides (where they are in contact with the aqueous phases and the polar head groups of the membrane lipids) which are separated by a hydrophobic belt. The latter is in contact with the alkyl chains of the lipids. As a result of this amphipathic nature of their surface, membrane proteins are not soluble in either aqueous or organic solvents. To isolate membrane proteins one first has to prepare the membranes, and then solubilize the membrane proteins by adding an excess of detergent. Detergents consist of a polar or charged head group and a hydrophobic tail. Above a certain concentration, the so-called critical micellar concentration (CMC), detergents form micelles by association of their hydrophobic tails. These micelles take up lipids. Detergents also bind to the hydrophobic surface of membrane proteins with their hydrophobic tails and form a ring-like detergent micelle surrounding the membrane protein, thus shielding the hydrophobic belt-like surface of the membrane protein from contact with water. This is the reason for their ability to solubilize membrane proteins, although with detergents with large polar head groups it is sometimes difficult to achieve a rapid and complete solubilization. The solubilizate, consisting of these mixed protein–detergent complexes as well as lipid-containing and pure detergent micelles, is then subjected to similar purification procedures as are soluble proteins. Of course, the presence of detergents complicates the purification procedures. The choice of the detergent is critical. The detergent micelles have to replace, and to mimic, the lipid bilayer as perfectly as possible, in order to maintain the stability and activity of the solubilized membrane protein. The solubilization of membrane proteins has been reviewed by Hjelmeland (1990[link]) and the general properties of the detergents used has been reviewed by Neugebauer (1990[link]).

References

Gerstein, M. (1998). Patterns of protein-fold usage in eight microbial genomes: a comprehensive structural census. Proteins, 33, 518–534.
Hjelmeland, L. M. (1990). Solubilization of native membrane proteins. Methods Enzymol. 182, 253–264.
Neugebauer, J. M. (1990). Detergents: an overview. Methods Enzymol. 182, 239–253.








































to end of page
to top of page