Porin trimers

Despite all the shortcomings listed above, full particle classical MD can be considered mature [84]. Even when all shortcomings will be overcome, we can now clearly delineate the limits for application. These are mainly in the size of the system and the length of the possible simulation. With the rapidly growing cheap computer memory shear size by itself is hardly a limitation several tens of thousands of particles can be handled routinely (for example, we report a simulation of a porin trimer protein embedded in a phospholipid membrane in aqueous environment with almost 70,000 particles [85] see also the contribution of K. Schulten in this symposium) and a million particles could be handled should that be desired. 

FIGURE 10.29 Three-dimensional recon-strnction of porin from Rhodohacter capsulatus. Drawings of (a) side view of porin monomer showing /3-sheet strnctnre. (b) Top view and (c) nearly top view of porin trimer. 

The outer membrane of Gram-negative bacteria is spanned by porins, trimeric proteins that form inlet channels between the outer membrane and the periplasmic space. The geometry of the porins defines the substrate uptake limit, which is on the order of 600 daltons (Weiss et al., 1991), approximately equivalent to a trisaccharide. Substrates larger than this limit (with few exceptions) must be hydrolyzed outside the outer membrane prior to uptake. Extracellular hydrolysis is carried out by means of extracellular en-... 

Besides porin trimers with 16- and 18-stranded /1-barrels, even larger 22-stranded /1-barrel proteins were found in the outer membrane, namely the monomeric active iron transporters FhuA and FepA. The lack of ATP or an equivalent energy carrier in the periplasm restricts the... 

Figure 3.29 Generalised Structure of Outer Membrane Porins. Although there is strong evidence that the porirvs exist in a trimeric configuration, the actual pore may exist as shown or be present Tn each individual porin molecule, i.e. 3 pores/porin trimer...

Each subunit of the trimeric porin molecule from R. capsulatus folds into a 16-stranded up and down antiparallel P barrel in which all p strands form... 

The complete porin molecule is a stable trimer of three identical subunits, three each with a functional channel (Figure 12.8). About one-third of the... 

Figure 4 (Plate 5). Atomic structure of the sucrose specific selective porin ScrY isolated from the outer membrane. Longer loops are directed to the outside, shorter turns are facing the periplasm. Monomer and assembled homotrimer in side view (left and middle) top view of assembled trimer (right). (Reproduced by permission of W. Welte and A. Brosig)...

Figure 8-20 MolScript ribbon drawings of the OmpF porin of E. coli. (A) View of the 340-residue monomer. (B) View of the trimer looking down the threefold axis. From Wa-tanabe et al3is From atomic coordinates of Cowan et al.3i9 (C) Molecular model of the constriction zone of the PhoE porin. Locations of key residues are shown, with positions of homologous residues in OmpF given in parentheses. Extracellular loops have been omitted. Constructed from coordinates of Cowan et al.349 by Samartzidou and Del-cour.342 Courtesy of Anne Delcour.

A model of the structure of porin from the outer membrane of Rhodobacter capsulatus. Part (a) shows the a-carbon backbones of a trimer of porin molecules viewed along an axis approximately perpendicular to the plane of the membrane. Each molecule forms a tube that passes across the membrane. Part (b) shows an individual porin monomer, enlarged slightly from (a) and viewed along an axis approximately in the plane of the membrane. The molecule folds as a jS-barrel with 16 antiparallel jS strands. (From M. S. Weiss, et al.. The three-dimensional structure of porin from Rhodobacter capsulatus at 3 A resolution, FEES Lett. 267 268, 1990. Copyright 1990 Elsevier Science Publishers BV, Amsterdam, Netherlands. Reprinted by permission.)... 

Figure 17.31 shows a model of porin from the outer membrane of the bacterium Rhodobacter capsulatus. Unlike most integral membrane proteins, porins have a j8-stranded secondary structure. The R. capsulatus protein appears to form a 16-stranded /3 barrel that crosses the membrane as a tube. The tubular molecules aggregate as trimers with three parallel pores. 

The behavior of such a large system as a pore formed by a bacterial porine (E. coli OmpF) has been simulated in a lipid bilayer of palmitoyloleoylphosphatidylethanola-mine (POPE) [95]. Despite the use of united atoms, the final system of the trimeric porin embedded into 318 POPE molecules and solvated with water consisted of more than 65 000 atoms in total. During the 1 ns of the MD simulation the trimeric structure remained stable, with almost all flexibility in the loops and turns outside the 3-strands. The movement and orientation of the water molecules was investigated in detail. As found in case of the pore formed by the hexameric LS3 helix bundle [90], the diffusion of the water was decreased to about 10% of that of bulk water. Some ordering of the water molecules was evident from the average water dipole moments, which showed a strong dependence on the vertical position within the porine. 

Fig. 1. Ribbon plot of the 16-stranded /bbarrel of the general porin from Rhodobacter capsulatus viewed from the molecular threefold axis (Weiss and Schulz, 1992). Note the large variation of the inclination angle a and the difference between the high barrel wall facing the membrane and the low wall at the trimer interface.

Channels are of course also formed by all porins. A general porin contains 16 /1-strands and has a shear number of 20 and a nearly circular cross section (Table II). Three parallel barrels associate to form trimers. The type of residues outlining the channel determines the specificity of a porin which, however, is usually not very strict. The two 18-stranded porins are very specific. Their channel cross-sections are actually smaller than those of the general porins in agreement with their higher selectivity. The 22-stranded barrels of the iron transporter proteins have circular cross sections and would form a very wide channel if they were not filled with the globular N-terminal 150-residue domain. 

On trimerization a nonpolar core is formed at the molecular threefold axis of the porins so that the central part of the trimer resembles a water-soluble protein. 

Seshadri, K., Garemyr, R., Wallin, E., von Heijne, G., and Elofsson, A. (1998). Architecture of /3-barrel membrane proteins analysis of trimeric porins. Protein Sci. 7, 2026-2032. 

The /3 sheet orientation in porin, a trimeric integral protein which forms channels across E.coli outer membranes, was determined by analyzing the amide I band and its shoulders around 1631 cm and the amide II band around 1530 cm. The results showed that the jS sheets are oriented perpendicularly to the membrane (Nabedryk et al., 1988). 

Figure 12.20. Structure of Bacterial Poriu (from Rhodopseudomonas blastica). Porin is a membrane protein built entirely of beta strands. (A) Side view. (B) View from the periplasmic space. Only one monomer of the trimeric protein is shown.

The porin monomers associate to form trimeric channels as is shown in Fig. 8-20B. They all have a central water-filled, elliptical channel that is constricted in the center to an "eye" -0.8 x 1.1 nm in size. In this restriction zone the channel is lined with polar residues that provide the substrate discrimination and gating. For example, in OmpF and FhoE there are many positively and negatively charged side chains that form the edge of the eye (Fig. 8-20C). The electrostatic potential difference across the outer membrane is small, but apparently determines whether the porins are in an open or a closed state. The voltage difference has opposite effects on OmpF and FhoE, apparently as a result of the differing distribution of charged... 

The outer membrane also contains a protein known as porin. The trimeric forms of porin form chaimels for the passage of small ionic molecules through the outer membrane. Other transport systems exist for higher molecular weight ionic substances. 

The amino acid sequences of porins are predominantly polar and contain no long hydrophobic segments typical of integral proteins with ct-helical membrane-spanning domains. X-ray crystallography has revealed that porins are trimers of identical subunits. In each subunit, 16 (3 strands form a barrel-shaped structure with a pore in the center (Fig-... 

A FIGURE 5-14 Structural model of one subunit of OmpX, a porin found in the E. coli outer membrane. All porins are trimeric transmembrane proteins. Each subunit Is barrel shaped, with p strands forming the wall and a transmembrane pore In the center. A band of aliphatic (noncycllc) side chains (yellow) and a border of aromatic (ring-contalning) side chains (red) position the protein In the bIlayer. [After G. E. Schulz, 2000, Curr. Opin. Struc. Biol. 10 443.]... 

Pori ns, pore-forming proteins originally discovered in the outer membrane of Gram-negative bacteria. The porins consist of trimers of identical subunits, and each subunit forms a 16-stranded anti-parallel /3-barrel containing a pore. They form aqueous channels used for the passive diffusion of water, ions and small molecules. Porins are found in bacterial cell walls, and also... 

Such mass spectrometric determinations of reactive sites in intact protein strac-tures by chemical modifications in proteins have been found highly useful in strac-ture—function studies of proteins [15], such as for ion-chatmel proteins (porins see Fig. 10.11). The structures of several bacterial porins have been determined by X-ray crystallography, such as the porin from Rhodobacter capsulatus (R. c.-porin) [132] which forms a trimeric complex of 16 18-stranded P-barrels. A characteristic stmc-ture element of R. c.-porin is a central constriction loop inside the P-barrel, which has been suggested as the central site determining cation/anion permeability and... 

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