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Protein Science (2002), 11:1309-1319.
Copyright © 2002 The Protein Society

Electrostatic properties of the anion selective porin Omp32 from Delftia acidovorans and of the arginine cluster of bacterial porins

Ulrich Zachariae1, Assen Koumanov2, Harald Engelhardt1 and Andrey Karshikoff2

1 Abteilung Molekulare Strukturbiologie, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany
2 Department of Bioscience at Novum, Karolinska Institute, S-14157 Huddinge, Sweden

Reprint requests to: Harald Engelhardt, Abteilung Molekulare Strukturbiologie, MPI für Biochemie D-82152 Martinsried bei München; e-mail: engelhar{at}biochem.mpg.de; fax: 49-89-85782641.

(RECEIVED December 11, 2001; FINAL REVISION February 22, 2002; ACCEPTED February 26, 2002)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.4910102.


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Conclusions
 Materials and methods
 References
 
The functional properties of the anion-selective porin Omp32 from the bacterium Delftia acidovorans, formerly Comamonas acidovorans, are determined by the particularly narrow channel constriction and the electrostatic field inside and outside the pore. A cluster of arginines (Arg 38, Arg 75, and Arg 133) determines the electrostatic field close to the constriction zone. Stacked amino acids carrying charges are prone to drastic pKa shifts. However, optimized calculations of the titration behavior of charged groups, based on the finite-difference Poisson-Boltzmann technique, suggest that all the arginines are charged at physiological pH. Protonation of the clustered arginines is stabilized by one buried glutamate residue (Glu 58), which is strongly interacting with Arg 75 and Arg 38. This functional arrangement of three charged amino acid residues is of general significance because it is found in the constriction zones of all known 16-stranded porins from the {alpha}-, ß-, and {gamma}-proteobacteria.

Keywords: Channel protein; outer membrane protein; Comamonas acidovorans; pKa calculations; charge cluster; ion selectivity; OmpF; PhoE


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Conclusions
 Materials and methods
 References
 
Porins are channel proteins that bridge the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts to allow the passage of hydrophilic solutes and ions (e.g., nutrients). In recent years, a number of bacterial outer-membrane proteins have been characterized by X-ray structural analysis. Porins have been found to form a ß-barrel structure consisting of 16 or 18 transmembrane ß-strands (Weiss et al. 1991; Cowan et al. 1992; Schirmer 1998). The siderophore-specific channels are built up from 22 ß-strands (Ferguson et al. 1998; Locher et al. 1998; Buchanan et al. 1999), and only 8 ß-strands form the barrel of the outer-membrane proteins belonging to the ß8-family (Baldermann et al. 1998; Vogt and Schulz 1999). Generally, porins form homotrimers in the bacterial outer membrane. One or more extracellular loops fold into the channel opening, constrict the channel cross-section, and contribute to the exclusion of bulky molecules and to the specific binding of ligands (e.g., with sugar-specific porins; Schirmer 1998; Koebnik et al. 2000). In particular the accumulation of charged residues in the constriction zone (i.e., the narrowest region of the pores), is assumed to have great influence on the ion conductance and selectivity properties of the porin channels (Weiss et al. 1991; Schirmer and Phale 1999).

Porin Omp32 from Delftia acidovorans, formerly Comamonas acidovorans (Wen et al. 1999), is a particularly interesting molecule for studying the electrostatic properties of the protein and the diffusion of inorganic and organic ions through the porin channels because it exhibits a number of unusual structural and functional features. (1) Omp32 exists as a heterohexameric molecule composed of the homotrimeric porin proper and an intimately associated homotrimeric peptide extending into the periplasmic space of the cell envelope (Zeth et al. 2000). (2) A conspicuous girdle of positively and negatively charged residues on the outer surface of the ß-barrel links the porin to the lipopolysaccharide head groups in the outer membrane. (3) Omp32 possesses the narrowest pore cross-section (~5 x 7 Å) of all structurally known porins (Zeth et al. 2000). (4) Omp32 shows a very high selectivity for anions (Mathes and Engelhardt 1998), and it is expected to be specific for organic acids, the preferred C-source of D. acidovorans. (5) Omp32 exhibits unusual functional properties in electrophysiological measurements, such as strongly asymmetric and nonlinear current-voltage curves (Mathes and Engelhardt 1998).

Along with the analysis of the atomic structure, the assessment of the electrostatic properties of the pore is a prerequisite to achieve insight into the mechanisms of ion conductance and ion selectivity of porins. Understanding the electrostatic and functional properties of Omp32 from D. acidovorans could also have some significance for the investigation of closely related porins from pathogenic microorganisms such as Bordetella pertussis, Neisseria gonorrhoeae, and Neisseria meningitidis, which are the causative agents for serious infectious diseases. A number of strains are assumed to have developed resistance against antibiotics due to mutations on their constriction-forming loop (Achouak et al. 2001).

In accord with other porins, such as OmpF and PhoE from Escherichia coli (Cowan et al. 1992), the constriction zone of Omp32 contains three conserved arginine residues in close spatial neighborhood. As opposed to other porin channels, the positive charges are not counterbalanced by acidic groups on the opposite channel wall in Omp32, a fact that was suggested to be one reason for the strong anion selectivity observed (Zeth et al. 2000). However, due to the close proximity of the three adjoining arginine side chains, these groups may manifest unusual ionization behavior leading to deprotonation of the cluster around neutral pH (Karshikoff et al. 1994) and to effects on the efficiency of ion selectivity.

Our study analyzes the titration behavior of ionizable groups and the electrostatic properties of porin Omp32, with a special emphasis on the arginine cluster close to the constriction zone. Our calculations suggest that these arginines are charged at physiological pH and reveal an arrangement of conserved amino acid residues that stabilize these charges in Omp32 and other 16-stranded porins.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Conclusions
 Materials and methods
 References
 
Omp32 possesses two functionally important regions with a high density of charged residues (i.e., the channel with the constriction zone and the lysine or charge girdle at the exoplasmic surface of the protein [Fig. 1Go; Zeth et al. 2000]). Because the charge status of these residues is of functional significance, we analyzed their electrostatic interactions by means of calculations based on a finite-difference Poisson-Boltzmann (FDPB) procedure (Karshikoff 1995; Altobelli et al. 2001; Koumanov et al. 2001). For our calculations, the truncated trimer model was used (i.e., monomeric structures with the addition of adjacent residues from the neighboring two subunits, including the latching loop L2 in the case of OmpF and PhoE; Karshikoff et al. 1994).



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  		Fig. 1. Regions with high density of charged amino acid residues in the anion selective porin Omp32. (A) View from the exoplasmic side of the porin. The cluster of charged amino acids inside the channel consists of three arginines and two glutamates situated opposite loop L3. L3 narrows the pore together with loop L8 and a protrusion emerging from ß-strand ß2. The charge girdle at the exoplasmic face of the porin mainly contains lysine and aspartic acid residues. (B) Side view of the charge cluster close to the constriction zone of the channel. The conformation of loop L8 is stabilized by a salt bridge between Lys 308 (K308) and Glu 60 (E60). Glu 58, forming salt bridges to Arg 75 and Arg 38, is a functional constitutent of the arginine cluster. Note that the glutamate residues do not protrude into the channel lumen. The figure was produced with DINO (http://www.biozentrum.unibas.ch/~xray/dino).

 
Salt bridges with structural significance in Omp32
Our calculations revealed a number of salt bridges that are involved in the structural stabilization of Omp32, in addition to hydrogen bridges that were described previously (Zeth et al. 2000). The most important ion-pair interactions occur between Glu 60 and Lys 308, Arg 92 and Asp 128, and Lys 100 and Asp 179. Lys 308 is located close to the tip of loop L8 that folds toward the pore and contributes to the channel constriction (Fig. 1Go). The charged groups of Glu 60 and Lys 308 have a distance of only 2.7 Å, explaining the large interaction energy (Table 1Go). The other two salt bridges stabilize the folding of loop L3 inside the pore. The electrostatic attraction between Lys 100 and Asp 179 immobilizes the {alpha}-helix portion of L3 in the periplasmic half of the pore. Similarly, the couple Arg 92–Asp 128 stabilizes the position of the protrusion of L3 (i.e., protrusion 2 according to Zeth et al. [2000] in the exoplasmic pore opening).


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  		Table 1. Charged amino acid residues and electrostatic interactions in porin Omp32 from Delftia acidovorans
 
Lysine belt of Omp32
A markedly polar region of ionizable residues located on the membrane-flanking outer wall of the ß-barrel structure, pointing toward the lipopolysaccharide head groups, is built up from 10 lysine residues and one arginine group surrounded by 10 aspartates and 1 glutamate (Fig.1).

The calculations showed that eight of the basic groups are fully protonated around physiological pH (Table 1Go), and three are largely protonated. All acidic groups around the lysine girdle were found to be ionized at pH 7. It has already been shown that the charged form of aspartic and glutamic acids is stabilized by backbone and side-chain dipoles that compensate for the desolvation penalty (Lancaster et al. 1996; Spassov et al. 1997; Gunner et al. 2000). A number of Lys–Asp pairs give rise to significant pKa shifts stabilizing the charged status over a large pH range.

Although the lysine girdle is flanked by an equal number of negative groups, the resulting electrostatic potential toward the membrane is positive (see below). The reason for this mainly lies in the architecture of the girdle, in which the lysine {varepsilon}-amino groups are exposed to the lipopolysaccharide head groups, and the aspartate carboxyl groups are more confined to the protein surface (Fig.1Go). The aspartates apparently serve as a factor stabilizing the charged form of the lysines with only limited influence on the electrostatic field surrounding the girdle. This could explain why the high amount of aspartic acids in this region of the molecule remained stable in evolution (i.e., has not been replaced by glutamate). A remarkable fact is that the charge girdle of the Rhodobacter blasticus porin (and of related porins) is almost exclusively built from negatively charged groups (Kreusch and Schulz 1994), with implications for the interaction with the lipopolysaccharides, which should be different in cells of the respective phylogenetic trees (i.e., of the ß- and {alpha}-subdivision of proteobacteria).

Constriction zone of Omp32
The constriction zone of the pore is the functionally important region where ions are selected for their size and partly for their charge. Omp32 contains three arginine residues close to the constriction zone (i.e., Arg 38, Arg 75, and Arg 133; Fig. 1BGo) that are referred to as the arginine cluster. In Omp32, no further ionizable residues are present in the channel constriction that could give rise to a transverse electric field as was observed for other porins (Weiss et al. 1991; Cowan et al. 1992; Dutzler et al. 1999). Despite the fact that the arginine residues are separated by distances of only 3.5 Å (Arg 38-N{eta}1–Arg 75-N{eta}1) and 3.37 Å (Arg 75-N{eta}2–Arg 133-N{eta}2) in the crystal structure, all of the three titratable groups were calculated to be fully protonated at pH 7 (Table 1Go). The calculations even suggested that Arg 75 remains protonated up to pH 12 and the flanking Arg 38 and Arg 133 to ~pH 8–9 (Fig. 2AGo).



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  		Fig. 2. (A) Calculated titration curves for side chains in the arginine cluster of Omp32. The guanidinium groups are almost fully protonated up to pH 9. (B) Potential energy of a negative elementary charge along the longitudinal axis of the porin channel aligned such that it is centered in the free lumen of the channel constriction zone (continuous line). The accessible pore cross-section of the trimeric porin (broken line) was calculated for a particle of radius 1.5 Å (water molecule) using program CROSS (A. Philippsen, Biozentrum Basel). The distance along the Z-axis is measured from the periplasmic rim of Omp32 toward the exoplasmic side.

 
The moderate extent of charge destabilization is partly due to the relatively low desolvation penalties (Born energies) for the arginine side chains (that protrude into the pore) in those cases where calculations were performed with a high-permittivity constant ({varepsilon} = 78) for the channel interior. Theoretical considerations suggest effective values ~{varepsilon} = 40 for different kinds of channels (Partenskii and Jordan 1992; Sansom et al. 1997) or confinement of water in nanocavities (Senapati and Chandra 2001). We performed calculations with Omp32 using values of {varepsilon} = 40, 30, and 20 to evaluate to what extent the protonation of the clustered arginines is dependent on the channel permittivity. At neutral pH the overall protonation of the three arginines is 97% ({varepsilon} = 40), 92% ({varepsilon} = 30), or 76% ({varepsilon} = 20), respectively. Protonation on Arg 133 appears to be most sensitive with respect to the effective value used; the average protonation amounts to 94% ({varepsilon} = 40), 83% ({varepsilon} = 30), or 60% ({varepsilon} = 20), respectively, whereas Arg 75 remains fully protonated.

The calculations revealed that the stabilization of the protonated states of Arg 75 and Arg 38 is largely due to the interaction of their guanidinium groups with the Glu 58 carboxylate and is supported by an additional but minor contribution from Glu 60. Glu 58 and Glu 60 are located on ß-strand 3 in a position backing the arginine groups (Fig. 1Go). At pH 7, the interaction energy between the charges on Arg 75 and Glu 58 amounts to -7.7 kcal/mole (Table 1Go) and increases if values lower than {varepsilon} = 78 are applied in the pore. The protonation of Arg 38 is stabilized by the negative charge of the Glu 58 side chain by -3.4 kcal/mole (Table 1Go), showing that especially the central arginine residue benefits from the interaction with Glu 58. As a result we found that the interaction with negative charges of acidic groups, but especially with Glu 58, accounts for the stabilization of the positive charges of the arginine cluster in Omp32. The accumulation of positive charges leads to a pronounced potential inside the pore.

Electrostatic potential of Omp32
The potential map of Omp32 (Fig. 3Go) shows that the influence of the three positive charges of the arginine cluster and the distribution of other positively charged residues in the channel lend the entire pore region a positive electrostatic potential. Along the longitudinal axis of the channel from the extracellular to the periplasmic entrance, the potential energy is highest ~5 Å below the minimum area of the constriction zone, that is, in the vicinity of the central Arg 75, where it amounts to approximately -3.6 kcal/mole for a unit negative charge (approx. -6.1 kT; Fig. 2BGo). The distinct anion selectivity of Omp32, with a selectivity factor around 20 (Mathes and Engelhardt 1998), can largely be attributed to the following structural and electrostatic peculiarities. (1) The geometry of the Omp32 channel constriction prevents that the electrostatic field, mainly originating from a particular area (i.e., the arginine cluster), fades significantly across the cross-section of the channel (Fig. 3Go). The latter is as small as 25 Å2, probed with a test molecule of 3 Å in diameter (Fig. 2BGo), and is considerably smaller than the cross-sections of other porins (H. Engelhardt, unpubl.). (2) There are no negatively charged residues that create an electric field across the channel and that could attenuate the positive potential in the constriction zone of Omp32. If negative charges were located at the opposite channel wall they would have a significant effect as was observed by comprehensive mutant studies with OmpF. The ion selectivity of OmpF changed by a factor of 4.5 if the two negatively charged residues, creating the cross-field together with the arginine cluster, were replaced by their amides (Phale et al. 2001). (3) The potential in the exoplasmic and periplasmic halves of the pore is positive throughout the channel (Figs. 2, 3GoGo). A number of basic residues contribute to that potential in addition to the arginine cluster (Table 1Go). Attraction of ions by the field at the pore entrances and selection of ions in the charge filter inside the channel are synergistic processes enhancing the anion selectivity of Omp32. This situation is different in OmpF, for instance, where a negative potential at the pore opening and the arginine cluster in the constriction zone do not cooperate and diminish the efficiency of cation selection. The cation selectivity of OmpF increased by a factor of ~12 if the clustered arginines were exchanged by uncharged amino acids (Phale et al. 2001).



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  		Fig. 3. Electrostatic potential of porin Omp32 calculated at pH 7 and an ionic strength of 0.12 M. The size and the color of the grid dots characterize the potential at the respective points (blue, positive; red, negative). The upper threshold value was set to 2.5 kcal/mole (represented by the largest dots). Views are from the exoplasmic face(A), periplasmic face(B), longitudinal section showing the inner half of the porin channel with the arginine cluster in the constriction zone(C), and longitudinal section oriented toward the lipid membrane(D). The strong positive potential in the top regions of A and B indicates the lysine girdle, the negative potential (bottom) the trimer contact regions. The figure was produced with DINO (http://www.biozentrum.unibas.ch/~xray/dino).

 
The extracellular region of Omp32 exhibits an isosurface of the electrostatic potential being shaped like a twofold funnel centered on the pore entrance (Fig. 4Go). It is easily imaginable that this funnel could lead anions from the surrounding environment toward the entrance of the channel. It is probable, however, that the shape of the isosurface near the trimer contact region will be modified if complete trimers are used for calculations. The only remarkable region with a negative potential in Omp32 is identical with the trimer contact region (Fig. 3Go). However, the contact site encloses complexed Ca2+ in vivo and is largely masked in the native porin (Zeth et al. 2000).



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  		Fig. 4. Isosurface of the electrostatic potential of Omp32, calculated for conditions of pH 7 and an ionic strength of 0.12 M. View from the exoplasmic face. The isosurface was drawn just beyond the level of zero, leaving the regions with positive potential free from the isosurface network. Two positive funnel-shaped regions direct anions from the environment toward the channel. The figure was produced with DINO (http://www.biozentrum.unibas.ch/~xray/dino).

 
The arginine cluster from proteobacterial porins
The insight that glutamate residues stabilize the protonation of the arginine cluster in Omp32 led us to examine homologous porin structures with respect to similar arrangements of basic and acidic groups. The arginine cluster is a conserved structure in porins; Arg 42, Arg 82, and Arg 132 represent the corresponding positions in OmpF and PhoE (Cowan et al. 1992) and Arg 9, Arg 24, and Arg 26 in the Rhodobacter capsulatus porin (Weiss and Schulz 1992). (It should be noted that in PhoE the true sequence positions are Lys 16, Lys 18, Arg 37, Arg 75, Lys 125, and Arg 126. Because of the position numbering in the original coordinate file [1PHO in the PDB data bank; Cowan et al. 1992], which was obviously adapted to the equivalent sequence positions of OmpF, the numbering appears identical for OmpF and PhoE. These sequence positions were also used in other studies and only for reasons of compatibility we refer to the artificial position numbering in the following text.) In all these porins the arginine cluster is extended by further positively charged residues (Fig. 5Go). In OmpF and PhoE, Glu 62 is located at a similar position relative to Arg 82 as Glu 58 is to Arg75 in Omp32. Here again the salt bridge between Arg 82 and Glu 62 leads to the stabilization of the protonated state of the central Arg 82 in both channel proteins. In the porin of R. capsulatus Asp 7 provides the stabilizing negative charge, here mainly interacting with Arg 26 and less strongly with the central Arg 24. The interaction energies are of similar size in each case (OmpF Glu 62–Arg 82: -8.1 kcal/mole; PhoE Glu 62–Arg 82: -8.3 kcal/mole; R. capsulatus porin Asp7–Arg26: -7.1 kcal/mole) and are comparable to that observed in Omp32 according to our calculations. This ion pair has been shown to be stable for 1.3 nsec in molecular dynamics simulations of OmpF (Suenaga et al. 1998). It should be noted that in the R. blasticus and Paracoccus denitrificans porins a tyrosine appears to play a role in charge stabilization instead (data not shown).



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  		Fig. 5. Architecture of the arginine clusters in the channel constrictions of porins OmpF (A), PhoE (B), and of the Rhodobacter capsulatus porin (C). The arginine clusters are extended by lysine residues in these porins. Glu 62 and Glu 71, the latter originating from loop L2 of the neighboring subunit, form salt bridges to Arg 82 and Arg 132/Arg 100 in OmpF and PhoE (A, B). In the porin of R. capsulatus Asp 7 interacts with Arg 26 of the arginine cluster, which represents the functional equivalent to the salt bridge of Arg 75 and Glu 58 in Omp32 or of Arg 82 and Glu 62 in the porins displayed in A and B. The figure was produced with DINO (http://www.biozentrum.unibas.ch/~xray/dino).

 
The effects of partial charges may induce drastic pKa shifts (Lancaster et al. 1996; Gunner et al. 2000) and are apparently of substantial importance for charge stabilization in porins. Previous studies showed that the protonation of the arginine cluster behaved less stable around neutral pH when partial charges were not included in the calculations (Karshikoff et al. 1994).

Another remarkable fact in OmpF and PhoE is the proximity of Glu 71 to Arg 100, as well as to the cluster-forming Arg 132. Glu 71 is situated on the latching loop L2 from the adjoining subunit that was included in our calculations. They revealed that the interaction energies between the pairs Glu 71–Arg 132 and Glu 71–Arg 100 are of comparable strength and that the salt bridge contributes to the stabilization of the protonated state of Arg 132 in both OmpF and PhoE. Control calculations not including the latching loop L2 from the neighboring subunit indeed yielded deprotonation above pH 8 (OmpF) or pH 6 (PhoE). The presence of auxiliary basic groups in the constriction zone of OmpF and PhoE apparently requires additional interactions with negatively charged residues for charge stabilization.

Experimental studies showed that the single channel conductance is largely independent of pH over the range from 4.3 to 9.4 for OmpF (Schirmer and Phale 1999) and from 6 to 10 for Omp34 from Acidovorax delafieldii, which is functionally a closely related porin to Omp32 (Brunen and Engelhardt 1995). More importantly, the ion selectivity is also hardly affected by titration between pH 5.5 and 8.5 in case of OmpF (Schirmer and Phale 1999) and between pH 6 and 9 for Omp34 (Brunen and Engelhardt 1995). These data could either denote that the titratable residues are not charged or that their charges are not titrated around physiological pH. Our calculations support the latter case. Chemical modification that removed the charges from arginine residues in the constriction zone of Omp34 showed the exceptional importance of these charges for anion selectivity: Upon modification, the porin became cation selective (Brunen and Engelhardt 1995). Corresponding results were reported for arginine mutants of the porin from P. denitrificans (Saxena et al. 1999). Exchange of one arginine (Arg 29 or Arg 31) resulted in attenuated anion selectivity, exchange of both the cluster arginines in the reversal of ion selectivity. These observations also support the view that the clustered arginine residues are charged.

The arginine cluster in porin sequences
If the role of Glu 58 in Omp32 and of the homologous residues in OmpF and PhoE is of general functional significance, equivalent residues should be found in related porins as well. Sequence alignments of porins from the ß- and {gamma}-subdivision of proteobacteria showed the highest similarities in the region from strand ß2 to strand ß4; porins from the {alpha}-subdivision do not align well and were disregarded here. Figure 6Go shows that the cluster forming Arg 38 (Arg 42) and Arg 75 (Arg 82) represent highly conserved amino acids being replaced only by lysines in few porins. The interesting finding is that among the negatively charged residues only Glu 58 (Glu 62) is invariable in the sequences listed and is neither replaced by aspartate nor by other amino acids, which corroborates its crucial role for the arginine cluster. This conclusion also holds for Glu 71 in porins of the {gamma}-group of proteobacteria.



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  		Fig. 6. Alignment of the ß2–ß4 region of porin sequences from bacteria belonging to the ß- and {gamma}-subdomain of the proteobacteria. Highly conserved charged residues are color-coded; residues belonging to the charge cluster in the constriction zone are in italics. The charged residue at position 42 (46) is located in the periplasmic pore entrance. Glu 45 (magenta) is located in the trimer contact region; Glu 58 (Glu 62), colored in red, and Glu 71 in the {gamma}-group of porins (red) form salt bridges with residues of the arginine cluster. The underlined sequence portions indicate amino acids forming ß-strands in the structurally solved porins. Amino acids located in loop L2 were not aligned with respect to the location of gaps. *Synonymous with Comamonas acidovorans, #Sequence numbering according to the coordinate (pdb) files of the Protein Data Bank.

 

   Conclusions
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusions
Materials and methods
 References
 
The three arginines in the channel constriction of bacterial porins create a strong potential that largely determines the anion selectivity in Omp32. In other bacterial porins the cluster is extended by further basic groups, which, together with negative charges at the opposite channel wall, form a strong transverse electrostatic field and modulate the ion selectivity of the channel. The charges in the porin constriction zone contribute, together with the charged head groups of the lipopolysaccharides in the outer membrane, to the exclusion of apolar substances from the periplasm that would affect the integrity of the cytoplasmic membrane otherwise. These functions are realized most effectively if the arginines located in close proximity are completely charged. The present study suggests that this is indeed the case in Omp32 and in related porins. A glutamate residue found in the sequences of all porins of the ß- and {gamma}-subdivision of proteobacteria and an aspartate in porins of the {alpha}-subdivision, plays the central role in stabilizing the charges on the positive cluster. Evolutionary stable arrangements of amino acids are usually of functional significance—here in the creation and stabilization of a strong electrostatic field inside an open channel. It should be noted that our findings are also of importance for theoretical studies that significantly depend and rely on the electrostatic data as a basis (e.g., modeling of ion flow through the porin channels).


   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusions
 Materials and methods
References
 
The calculations presented in this work are based on a continuum electrostatic model. All protein partial charges were included in the atomic model. A thorough description of the computation of the protonation/deprotonation equilibrium of titratable sites is given elsewhere (Karshikoff 1995; Altobelli et al. 2001; Koumanov et al. 2001). In all calculations the bacterial outer membrane was simulated as infinite layer surrounding the porin monomer perpendicular to the longitudinal axis of the pore (Karshikoff et al. 1994). The membrane layer with entire thickness of 36 Å was composed of three sublayers. The central one represents the aliphatic membrane interior and is characterized by a dielectric constant of 4. It is situated between two sublayers with a higher permittivity of 40, representing the polar-head groups of the membrane lipids and lipopolysaccharides. The thickness of the polar sublayers was set to 10 Å and 7 Å at the extracellular and periplasmic side, respectively. A dielectric constant of 4 was assigned to the protein moiety. The periplasm and the cell exterior, as well as the pore interior, were modeled as solvent with a dielectric constant of 78 (Hamer 1971) and an ionic strength of 0.12 M. As far as the electrostatic properties of the water inside a narrow channel are expected to differ from that of the bulk (Partenskii and Jordan 1992; Sansom et al. 1997), additional calculations were performed with pore permittivities of 40, 30, and 20. Partial atomic charges were taken from the CHARMM22 parameter set (MacKerell et al. 1998). The atomic radii were taken from Rashin et al. (1986). The X-ray structure of Omp32 (PDB entry 1E54; resolution 2.1Å; Zeth et al. 2000), OmpF and PhoE (PDB entries 2OMF and 1PHO, respectively; resolution 2.4 Å and 3 Å, respectively; Cowan et al. 1992), and the R. capsulatus porin (PDB entry 2POR; resolution 1.8 Å; Weiss and Schulz 1992) were used. All hydrogen positions were obtained by the HBUILD routine of CHARMM (Brünger and Karplus 1988).

The linearized Poisson-Boltzmann equation was solved by a finite difference method in cubic grid boxes (96 x 96 x 96) with a grid spacing of 0.94 Å. The final values for the strongest charge–charge interactions and the desolvation energy were refined by two subsequent focusing steps on each ionizable group with a grid spacing of 0.42 Å and 0.21 Å, respectively.


   Acknowledgments
 
We thank Ansgar Philippsen for his program CROSS, we are grateful to Rudolf Ladenstein for his support, and we thank our colleagues Thomas Klühspies and Erik Roth for stimulating discussions. The project was supported by a grant of the Deutsche Forschungsgemeinschaft (En 144/3–1).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Conclusions
 Materials and methods
 References
 
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Baldermann, C., Lupas, A., Lubieniecki, J., and Engelhardt, H. 1998. The regulated outer membrane protein Omp21 from Comamonas acidovorans is identified as a member of a new family of eight-stranded ß-sheet proteins by its sequence and properties. J. Bacteriol. 180: 3741–3749.[Abstract/Free Full Text]

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