Properties of chemically modified porin from Escherichia coli in lipid bilayer membranes

Purified porin OmpF from Escherichia coli outer membrane was chemically modified by acetylation and succinylation of amino groups and by amidation of the carboxyl groups. Native and chemically modified porins were incorporated into lipid bilayer membranes and the permeability properties of the pores were studied. Acetylation and succinylation of the porin trimers had almost no influence on the single channel conductance in the presence of small cations and anions and the cation selectivity remained essentially unchanged as compared with the native porin. Amidation had also only little influence on the single channel conductance and changed the pore conductance at maximum by less than 50%, whereas the cation selectivity of the porin is completely lost after amidation. The results suggest that the structure of the porin pore remains essentially unchanged after chemical modification of the pores and that their cation selectivity is caused by an excess of negatively charged groups inside the pore and/or on the surface of the protein. Furthermore, it seems very unlikely that the pore contains any positively charged group at neutral pH.     

X-ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from Rhodobacter capsulatus: implications for ion selectivity and single-channel conductance.

The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinylporins determined by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ionization mass spectrometry identified selective succinylation of three lysine-epsilon-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal alpha-amino group. The structure of a tetra-succinylated porin (TS-porin) was determined to 2.4 A and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di-succinylated porin (MS-porin) and a TS-porin are localized at the inner channel surface and are solvent-accessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic entrance of the channel. The Lys-46 residue at the central constriction loop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS-and TS-porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single-channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure-selective modification of charged residues as an efficient approach in the structure-function evaluation of ion channels, and X-ray crystallography and mass spectrometry as complementary analytical tools for defining precisely the chemically modified structures.     

Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins.

Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E). All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius. Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner. The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel. The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much. These results suggest that PhoE specializes in the uptake of negatively charged solutes. At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge. However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size. This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels.     

The mechanism of ion selectivity of OmpF-porin pores of Escherichia coli

The OmpF porin from the outer membrane of Escherichia coli acts as a lightly cation-selective pore, allowing the diffusion of small cations and cationic molecules, whose Mr are a little larger than the threshold exclusion limit. To ascertain the mechanism of this cation selectivity, we have examined a possible influence of cationic solutes on the fluorescence emission and the circular dichroic spectrum of tryptophan residues of the porin trimer, searching for conformational change(s). The diffusion of cationic solutes was determined with the native and the amidated porins in the presence or the absence of the effector cations. The following results were obtained. (a) Cations, e.g. spermidine, caused fluorescence quenching in the native trimer, with a half-maximum fluorescence quenching at 11-18 microM. A change in the circular dichroic spectrum was also recorded at around 280 nm. (b) The dissociation constant of spermidine to the native trimer was calculated to be 16 microM as determined by the method of equilibrium dialysis. (c) The cation-caused fluorescence quenching was reversed when the carboxyl groups of the trimer were modified by the amidation reaction, though amidation of the trimer resulted in no significant change in the fluorescence intensity. (d) The diffusion rate of N-benzyloxycarbonyl-glycyl-L-prolyl-L-arginine p-nitroanilide through the native and the amidated porins was lowered in the presence and the absence, respectively, of cations. Both the extent of fluorescence quenching in the presence of cation and the rate of cation diffusion were inversely proportional to the number of amidated carboxyl residues. The relative fluorescence quenching of the porin trimer (the amidated versus the native) in the presence of cations was linearly related to the relative solute diffusion via the porin (the amidated versus the native). These results suggested that cations caused a conformational change in the trimer, resulting in an easier diffusion of the solutes. The results suggested further that a limited number of carboxyl groups in the pore interior are involved in the cation selectivity of OmpF-porin pores.     

Redistribution of the electric field within the pore contributes to the voltage-dependence of mitochondrial porin channel.

The effects of pH on the integral conductance and on the properties of single channels induced by porin from rat liver mitochondria in a lipid bilayer have been studied. When the membrane potential increases, the conductance of the multi-channel membrane decreases more sharply at acidic pH than at neutral or basic pH. The channel is shown to have several states with different conductance and selectivity. The number of levels and their conductance do not depend on pH, while the selectivity as well as the dependence of steady-state probabilities of different levels on the membrane potential are substantially affected by a pH change. This dependence curve steepens in the pH region where charges of carboxyl groups of aspartic and glutamic amino acids are neutralized. It is concluded that at neutral pH the channel gate is controlled by a great number of the positively and negatively charged groups. The high steepness of the conductance-voltage curve in the acidic region suggests that at least 60 positive charges participate in controlling the channel gate. This number, compared with that of the positively charged side chain amino acids per channel, according to the amino acid analysis of the porin, led us to conclude that almost all amino groups of the channel former must pass through the entire membrane potential difference upon random motion of the channel among the states. The assumption that channel closing leads to redistribution of the electric field within the pore, changing the energy of the charges on the voltage sensor, may be the only explanation of this phenomenon.     

Accessibility of lysyl residues of Escherichia coli B/r porin (OmpF) to covalent labeling reagents of different sizes. An approach for a three-dimensional structure of a channel-forming protein

The three-dimensional structure of Escherichia coli B/r porin (OmpF) was studied by chemical modification using activated sugars of different size. Galactose and galactosides of different penetration properties through the porin channel were oxidized by galactose oxidase, and the 6-aldehydes formed were linked to amino groups in porin by reduction with NaBH3CN. Tryptic fragments of modified and unmodified porin were separated by reversed-phase high pressure liquid chromatography and identified by amino acid and amino-terminal analysis from the known primary structure of OmpF. Modification of purified native porin trimers in beta-octylglucoside revealed three classes of amino groups: (i) those not modified by any sugars; (ii) those modified only by small sugars that diffuse rapidly through the pore, such as galactose or melibiose; and (iii) those modified by either small or large sugars, the latter including pore-impermeant sugars such as stachyose. The results suggest that the three classes of amino groups correspond, respectively, to groups buried in the trimeric molecule, those in the interior of the pore and those exposed on the surface of porin. In addition modification experiments performed on whole cells suggested that all the reactive groups modified by the pore-impermeant sugars (class iii) are located on the surface of porin exposed on the outside of the outer membrane.     

Effects of pore mutations and permeant ion concentration on the spontaneous gating activity of OmpC porin

Porins are trimers of beta-barrels that form channels for ions and other hydrophilic solutes in the outer membrane of Gram-negative bacteria. The X-ray structures of OmpF and PhoE show that each monomeric pore is constricted by an extracellular loop that folds into the channel vestibule, a motif that is highly conserved among bacterial porins. Electrostatic calculations have suggested that the distribution of ionizable groups at the constriction zone (or eyelet) may establish an intrinsic transverse electrostatic field across the pore, that is perpendicular to the pore axis. In order to study the role that electrostatic interactions between pore residues may have in porin function, we used spontaneous mutants and engineered site-directed mutants that have an altered charge distribution at the eyelet and compared their electrophysiological behavior with that of wild-type OmpC. We found that some mutations lead to changes in the spontaneous gating activity of OmpC porin channels. Changes in the concentration of permeant ions also altered this activity. These results suggest that the ionic interactions that exist between charged residues at the constriction zone of porin may play a role in the transitions between the channel's closed and open states.     

Porin channels in Escherichia coli: studies with beta-lactams in intact cells

Wild-type Escherichia coli K-12 produces two porins, OmpF (protein 1a) and OmpC (protein 1b). In mutants deficient in both of these "normal" porins, secondary mutants that produce a "new" porin, protein PhoE (protein E), are selected for. We determined the properties of the channels produced by each of these porins by measuring the rates of diffusion of various cephalosporins through the outer membrane in strains producing only one porin species. We found that all porin channels retarded the diffusion of more hydrophobic cephalosporins and that with monoanionic cephalosporins a 10-fold increase in the octanol-water partition coefficient of the solute produced a 5- to 6-fold decrease in the rate of penetration. Electrical charges of the solutes had different effects on different channels. Thus, with the normal porins (i.e., OmpF and OmpC proteins) additional negative charge drastically reduced the penetration rate through the channels, whereas additional positive charge significantly accelerated the penetration. In contrast, diffusion through the PhoE channel was unaffected by the presence of an additional negative charge. We hypothesize that the relative exclusion of hydrophobic and negatively charged solutes by normal porin channels is of ecological advantage to E. coli, which must exclude hydrophobic and anionic bile salts in its natural habitat. The properties of the PhoE porin are also consistent with the recent finding (M. Argast and W. Boos, J. Bacteriol. 143:142-150, 1980; J. Tommassen and B. Lugtenberg, J. Bacteriol. 143:151-157, 1980) that its biosynthesis is derepressed by phosphate starvation; the channel may thus act as an emergency pore primarily for the uptake of phosphate and phosphorylated compounds.     

Outer membrane porin protein of Haemophilus influenzae type b: pore size and subunit structure

The 40-kDa porin protein of Haemophilus influenzae type b was reconstituted into proteoliposomes. The relative rates of diffusion of small uncharged sugars across the channels formed by this protein were determined by measuring the rates of osmotic swelling of the liposomes. From these rates, a pore diameter of 1.8 nm was estimated using the Renkin equation. A chemical cross-linking technique was used to investigate the oligomeric structure of the 40-kDa porin. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis revealed the presence of porin dimers and trimers after reaction of the protein with dithio-bis-(succinimidyl propionate). These results confirmed that the porin of H. influenzae forms large water-filled channels and indicated that it probably exists as trimers in the outer membrane.     

The gate of mitochondrial porin channel is controlled by a number of negative and positive charges

Negatively charged carboxyl groups of mitochondrial porin have been converted into positively charged ones by means of reaction with water-soluble carbodiimide in the presence of ethylenediamine. Properties of channels formed in a planar lipid bilayer by native and modified porins are compared. Amidation has only little influence on the porin channel-forming activity as well as on the open-state conductance of the channel. However, the modification results in a significant enhancement of the voltage dependence of the channel gating and in an increase of the anionic selectivity. It is suggested that the voltage sensor of the porin channel gate is composed of a number of negative (greater than 14) and positive (greater than 22) charges.     

Relevance of charged groups for the integrity of the S-layer from Bacillus coagulans E38-66 and for molecular interactions.

In this paper, the importance of charged amino and carboxyl groups for the integrity of the cell surface layer (S-layer) lattice from Bacillus coagulans E38-66 and for the self-assembly of the isolated subunits was investigated. Amidination of the free amino groups which preserved their positive net charge had no influence on both. On the other hand, acetylation and succinylation, which converted the amino groups into either neutral or negatively charged groups, and amidation of carboxyl groups were accompanied by the disintegration or at least by the loss of the regular structure of the S-layer lattice. Treatment of S-layer monolayers with the zero-length cross-linker carbodiimide led to the introduction of peptide bonds between activated carboxyl groups and amino groups from adjacent subunits. This clearly indicated that in the native S-layer lattice the charged groups are located closely enough for direct electrostatic interactions. Under disrupting conditions in which the S-layer polypeptide chains were unfolded, 58% of the Asx and Glx residues could be amidated, indicating that they occur in the free carboxylic acid form. As derived from chemical modification of monolayer self-assembly products, about 90% of the lysine and 70% of the aspartic and glutamic acid residues are aligned on the surface of the S-layer protein domains. This corresponded to 45 amino groups and to 63 carboxyl groups per S-layer subunit. Labelling experiments with macromolecules with different sizes and charges and adsorption studies with ion-exchange particles revealed a surplus of free carboxyl groups on the inner and on the outer faces of the S-layer lattice. Since the carboxyl groups on the outer S-layer face were accessible only for protein molecules significantly smaller then the S-layer protomers or for positively charged, thin polymer chains extending from the surface of ion-exchange beads, the negatively charged sites must be located within indentations of the corrugated S-layer protein network. This was in contrast to the carboxyl groups on the inner S-layer face, which were found to be exposed on elevations of the S-layer protein domains (D. Pum, M. Sára, and U.B. Sleytr, J. Bacteriol. 171:5296-5303, 1989).     

Accessibility of the CLC-0 Pore to Charged Methanethiosulfonate Reagents

Using the substituted-cysteine-accessibility method, we previously showed that a cysteine residue introduced to the Y512 position of CLC-0 was more rapidly modified by a negatively charged methanethiosulfonate (MTS) reagent, 2-sulfonatoethyl MTS (MTSES), than by the positively charged 2-(trimethylammonium)ethyl MTS (MTSET). This result suggests that a positive intrinsic pore potential attracts the negatively charged MTS molecule. In this study, we further test this hypothesis of a positive pore potential in CLC-0 and find that the preference for the negatively charged MTS is diminished significantly in modifying the substituted cysteine at a deeper pore position, E166. To examine this conundrum, we study the rates of MTS inhibitions of the E166C current and those of the control mutant current from E166A. The results suggest that the inhibition of E166C by intracellularly applied MTS reagents is tainted by the modification of an endogenous cysteine, C229, located at the channel's dimer interface. After this endogenous cysteine is mutated, CLC-0 resumes its preference for selecting MTSES in modifying E166C, reconfirming the idea that the pore of CLC-0 is indeed built with a positive intrinsic potential. These experiments also reveal that MTS modification of C229 can inhibit the current of CLC-0 depending on the amino acid placed at position 166.     

Anti-HIV activity of a series of cosalane amino acid conjugates

The binding of the anti-HIV agent cosalane to CD4 is thought to involve ionic interactions of negatively charged carboxylates of the ligand with positively charged residues on the surface of the protein. The purpose of the present study was to examine the hypothesis that the two carboxyl groups of cosalane could be sacrificed through conjugation to amino acids, and the anti-HIV activity still be retained, provided that at least two new carboxyl groups are contributed by the amino acid residues.     

Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release.

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).     

The ligase chain reaction distinguishes hepatitis B virus S-gene variants

Abstract The functional significance of charged amino acids of the anion-selective porin Omp34 from Acidovorax delafieldii was investigated by means of conductance measurements. Chemical modification of Lys and Arg as well as titration of charges by adjusting the pH value revealed that positively charged amino acid residues determine the major functional properties of the porin. Positive charges are involved in creating the protein surface potential, the selectivity filter inside the channels, and the voltage-sensing and/or gating mechanisms.     

Comparison of secretory protein profiles in developing rat pancreatic rudiments and rat acinar tumor cells

Chemical modification of amino groups in matrix porin solubilized and purified from outer membranes of Escherichia coli in beta- octylglucoside was performed with eosin isothiocyanate and citraconic anhydride. At pH 7 8.5, the former reagent labeled a single amino group in the native protein, while more extensive derivatization was observed with increasing pH or upon denaturation. Citraconic anhydride modified approximately 12-14 residues in native porin and 15-16 of the total of 19 amino groups in the denatured state. Fluorescamine, another amine- specific reagent of intermediate size, derivatized 3 and 16 residues in the native and denatured states, respectively. These results indicate that reactive probes of various sizes may serve as indicators for the surface accessibility of reactive residues in matrix porin. The increased derivatization of lysyl residues at high pH (or in phosphate buffer) suggests the method's sensitivity to different conformational states of the protein. The extent of tyrosine modification (1-2 residues in the native, and approximately 22 in the denatured porin) depended on the state of protein folding, even with reagents of small size. The approach of using various probes with differing properties and specificities thus appears useful for the determination of membrane protein asymmetry, pore topology, and conformational states of transmembrane proteins.     

Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

To determine the contribution of charged amino acids to binding with the photosystem II complex （PSII）, the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N- succinimidyl propionate （NSP） or glycine methyl ester （GME） in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.     

Diffusion of beta-lactam antibiotics through oligomeric or monomeric porin channels of some gram-negative bacteria

The penetration of anionic beta-lactam antibiotics through porins was evaluated as a mechanism of drug resistance. The major proteins with porin activity were purified from the outer membranes of six bacteria. Three of the six porins were oligomeric porins. The molecular weights of their monomers were 37 kDa from Photobacterium damsela, 42 kDa from Serratia liquefaciens, and 36 kDa from E. coli B. The other three porins were heat-modifiable monomeric porins with molecular weights of 43 kDa from Porphyromonas asaccharolytica and Acinetobacter baumannii, and 37 kDa from Escherichia coli K12.Comparison of the six porin proteins revealed that, independent of their aggregation state, their amino acid content is similar but not identical. All have double the amount of negatively charged amino acids compared with positively charged amino acids. They have a similar polarity and polarity index. Two of the six tested bacteria do not produce beta-lactamase. These two bacteria were sensitive to the different beta-lactams tested. The other four bacteria were resistant to all or to several beta-lactams.A modified liposome swelling method was used for determining the rate of penetration of charged beta-lactam antibiotics. Zwitterionic beta-lactams were found to penetrate into liposomes at a rate that more or less fits their molecular weight, whether the porins are monomeric or oligomeric. The penetration rates of negatively charged beta-lactams are different for oligomeric and monomeric porins. Negatively charged beta-lactams penetrate through oligomeric porins better than estimated by their molecular weight, whereas monomeric porins are less penetrable to negatively charged beta-lactams than estimated by their molecular weight. The contribution of all types of porins to the susceptibility of bacteria to beta-lactam antibiotics (zwitterionic or negatively charged) is apparently doubtful. The porins may decrease or increase bacterial penetration rates to beta-lactams, and only the existence of a potential beta-lactamase that can destroy the penetrating drug will cause resistance.     

Chemical modification of low-density lipoprotein enhances the number of binding sites for divalent cations.

The EPR technique with paramagnetic Mn(II) ions has been used to probe the negatively charged sites on the surface of modified low-density lipoprotein (LDL). LDL modified in five different ways exhibited increased binding capacity for divalent cations. Enhanced binding is caused by the increase in the number of 'strong' binding sites. The 'strong' sites have been identified to be the aspartic acid and/or glutamic acid carboxyl residues and the 'weak' sites are zwitter-ionic phospholipids. In native LDL the negative groups make 'bonds' with the positive lysyl residues, thus stabilizing the structure. Any deprotonation or modification of the lysine amino groups makes the LDL structure more loose and the amino acid carboxyl groups accessible to divalent cations.     

猪链球菌2型溶血素的化学修饰

用DTT、H2O2、DEPC、EDC、NAI、NBS、PCMB、2,3Diacetyl和SA 等9种化学修饰剂,处理猪链球菌2型江苏分离株提纯的溶血素,研究其分子中氨基酸侧链基团与其溶血活性的关系。结果表明,巯基、氨基、羧基和酪氨酸残基等与其溶血活性无关,而色氨酸、组氨酸和精氨酸的化学修饰引起溶血活性的大幅度下降,二硫键的化学修饰引起溶血活性的大幅度增强。显示色氨酸、组氨酸和精氨酸残基是该溶血素的活性必需基团,二硫键的断裂可引起其活性增强。