franc pattus

The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore-forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.

Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and E1. These hybrid colicins were purified and their properties were studied. All of them were active against sensitive cells, although to varying degrees. From the results, one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin E1 is located in the N-terminal domain of colicin E1; (4) the 183 N-terminal amino acid residues of colicin E1 are sufficient to promote E1AA uptake and thus probably colicin E1 uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C-terminal domains of colicin A and colicin E1 by their respective immunity proteins.

A large number of mutations which introduce deletions in colicin A have been constructed. The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) was assayed. The receptor-binding properties of each protein were also analysed. From these results, we suggest that the NH2-terminal region of colicin A (residues 1 to 172) is involved in the translocation step through the outer membrane. The central region of colicin A (residues 173 to 336) contains the receptor-binding domain. The COOH-terminal domain (residues 389 to 592) carries the pore-forming activity.

Homogenous maltoporin (lamB protein), an Escherichia coli outer membrane spanning protein, was incorporated in phospholipid planar bilayers. It generates aqueous channels distinct from those formed by the non-specific porin (OmpF) or by phosphoporin (phoE protein). The single conductance, 150 pS in 1 M NaCl, is much smaller than that of the porins. The channels, which are poorly selective for cations and voltage independent, are specifically inhibited by maltose and maltodextrins. This inhibition, observed in the absence of maltose binding protein, demonstrates that the selectivity of maltoporin for maltose and maltodextrins is an intrinsic property of the protein.

The topology of the integral membrane protein Cai (colicin A immunity protein), which is required to protect producing cells from the pore-forming colicin A, was analysed using fusions to alkaline phosphatase. The properties of these fusion proteins support the model for Cai topology previously proposed on theoretical grounds. The protein was found to contain four transmembrane sequences and its N-and C-terminal regions were found to be directed towards the cytoplasm. Oligonucleotide-directed mutagenesis and sequence comparisons between Cai, Cbi (colicin B immunity protein), and Cni (colicin N immunity protein) were carried out to determine the functional regions of Cai. The possible roles of the various regions of Cai in its protective function and in its topological organization are discussed.

The dependence on pH and membrane potential of the pore formed by colicin A and its C-terminal 20 kDa fragment has been measured using planar lipid bilayers. The single channel conductance of the pore formed by both colicin A and the fragment increases with pH with an apparent pK of 6.0. At pH 5.0 the gating by membrane potential of the channels formed by either colicin A or its fragment is identical. At the same pH, quite similar pore properties were found when using the related bacteriocin, colicin E1. In agreement with previous studies, these data indicate that the protein structure containing the lumen of the pore resides in the 20 kDa C-terminal part of the colicin A and favours the recently proposed model, based on protein sequence analysis, which proposes that colicin A, E1 and IB C-terminal domains are folded in the same three-dimensional structure. However, it is also shown that colicin A and not its C-terminal fragment undergoes a pH dependent transition between an “acidic” and a “basic” form of the pore with an apparent pK of 5.3. The two forms of the pore differ by their gating charge but not by the channel size. These results suggest that there is a pH dependent association between the C-terminal domain carrying the lumen of the pore and another domain of the molecule which affect the pore sensitivity to membrane potential.

We have constructed, by recombinant DNA techniques, one hybrid protein, colicin A-beta-lactamase (P24), and two modified colicin As, one (P44) lacking a large central domain and the other (PX-345) with a different C-terminal region. The regulation of synthesis, the release into the medium and the properties of these proteins were studied. Only P44 was released into the medium. This suggests that both ends of the colicin A polypeptide chain might be required for colicin release. None of the three proteins was active on sensitive cells in an assay in vivo. However, P44 was able to form voltage-dependent channels in phospholipid planar bilayers. Its lack of activity in vivo is therefore probably caused by the inability to bind to the receptor in the outer membrane. PX-345 is a colicin in which the last 43 amino acids of colicin A have been replaced by 27 amino acids encoded by another reading frame in the same region of the colicin A structural gene; it was totally unable to form pores in planar bilayers at neutral pH but showed a very slight activity at acidic pH. These results confirm that the C-terminal domain of colicin A is involved in pore formation and indicate that at least the 43 C-terminal amino acid residues of this domain play a significant role in pore formation or pore function. Fifteen monoclonal antibodies directed against colicin A have been isolated by using conventional techniques. Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A. In addition, the altered forms of the colicin A polypeptide were used to map the epitopes of ten monoclonal antibodies reacting specifically with colicin A. Some of the antibodies did not bind to colicin A when it was pre-incubated at acidic pH suggesting that colicin A undergoes conformational change at about pH 4. The effects of monoclonal antibodies on activity in vivo of colicin A were investigated. The degree of inhibition observed was related to the location of the epitopes, with monoclonal antibodies reacting with the N terminus giving greater inhibition. The monoclonal antibodies directed against the C-terminal region promoted an apparent activation of colicin activity in vivo.

Conformational investigations, using circular dichroism, on the pore-forming protein, colicin A (Mr 60 000), and a C-terminal bromelain fragment (Mr 20 000) were undertaken to estimate their secondary structure and to search for pH-dependent conformational changes. Colicin A and the bromelain peptide are mainly alpha-helical with an enrichment of the alpha-helical content in the C-terminal domain carrying the ionophoric activity. The non-negligible beta-sheet structure in the C-terminal domain is unstable and is easily transformed into alpha-helix upon decreasing the polarity of the solvent. No evidence of pH-dependent conformational modification, correlated with modification of colicin A activity, could be obtained. The secondary structure estimated on the basis of experimental data favoured a model in which the pore is built of a minimal number of six transmembrane alpha-helical segments. Search for such segments in the amino acid sequence of the C-terminal domain of colicin A was carried out by combining secondary structure prediction methods with hydrophobicity and hydrophobic movement calculations. Similar calculations on the C-terminal domains of colicin E1 and IB indicate a common structure of the pores formed by colicin A, E1 and IB. Only two or three putative transmembrane segments could be selected in the sequences of colicin A, IB or E1. As a result, it is concluded that the channel is probably not built by a single colicin molecule but more likely by an oligomer.

Pseudomonas aeruginosa OprD is a 420-amino-acid protein that facilitates the uptake of basic amino acids, imipenem and gluconate across the outer membrane. OprD was the first specific porin that could be aligned with members of the non-specific porin super-family. Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD-topology model was proposed. Sixteen β-strands were predicted, connected by short loops at the periplasmic side. The eight external loops were of variable length but tended to be much longer than the periplasmic ones. Polymerase chain reaction (PCR)-based site-specific mutagenesis was performed to delete separately short stretches (4-8 amino acid residues) from each of the predicted external loops. The mutants with deletions in the predicted external loops L1, L2, L5, L6, L7 and L8 were tolerated in both Escherichia coli and P. aeruginosa. The expressed mutant proteins maintained substantial resistance to trypsin treatment in the context of isolated outer membranes. Proteins with deletions in loops L1, L5, L6, L7 and L8 reconstituted similar imipenem supersusceptibility in a P. aeruginosa OprD::Ω background. The L2-deletion mutant only partially reconstituted supersusceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.

The porins of Gram-negative bacteria are responsible for the ‘molecular sieve’ properties of the outer membrane. They form large water-filled channels which allow the diffusion of hydrophilic molecules into the periplasmic space. Owing to the strong hydrophilicity of their amino acid sequence and the nature of their secondary structure (beta strands), conventional hydropathy methods for predicting membrane topology are useless for this class of protein. The large number of available porin amino acid sequences was exploited to improve the accuracy of the prediction In combination with tools detecting amphipathicity of secondary structure. Using the constraints of β-sheet structure these porins are predicted to contain 16 membrane-spanning strands, 14 of which are common to the two (enteric and the neisserial) porin subfamilies.

The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin. The results of far- and near-UV circular dichroism measurements indicated that processing with trypsin does not lead to major changes in secondary or tertiary structure of the protein. An increase in tryptophan fluorescence intensity and a small red shift in the maximum emission wavelength of tryptophans could be observed, suggesting that there is a change in the environment of some of the tryptophans. There was also a dramatic increase in the binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalenesulfonate during activation, leading us to conclude that a hydrophobic region in the protein is exposed by trypsin treatment. Using measurements of light scattering, various parameters influencing oligomerisation of trypsin-activated aerolysin were determined. Oligomerization rates were found to increase with the concentration of aerolysin, whereas they decreased with increasing ionic strength.

Four outer-membrane proteins of Aeromonas hydrophila were purified and their N-terminal sequences and channel-forming properties were determined. Three could be matched with proteins from other species. One was a maltoporin, as its level increased when cells were grown in maltose-containing media, and the channel it formed was blocked by maltose. Another was like OmpF and OmpC of Escherichia coli, except that its channel fluctuated much more rapidly. The third protein, which was produced in low-phosphate medium, exhibited several properties of the general anion porin PhoE. The fourth showed no similarity to any known proteins. It had a unique N-terminus and it formed small sharply-defined cation-selective channels. Two other proteins which corresponded to OmpW of Vibrio cholerae and E. coli OmpA were partly characterized.

The cytolytic toxin aerolysin was found to form ion channels which displayed slight anion selectivity in planar lipid bilayers. In voltage-clamp experiments the ion current flowing through the channels was homogeneous indicating a defined conformation and a uniform size. The channels remained open between −70 to +70 mV, but outside this range they underwent voltage-dependent inactivation which was observed as open-closed fluctuations at the single-channel level. Zinc ions not only prevented the formation of channels by inhibiting oligomerization of monomeric aerolysin but they also induced a closure of preformed channels in a voltage-dependent fashion. The results of a Hill plot indicated that 2–3 zinc ions bound to a site within the channel lumen. Proaerolysin, and a mutant of aerolysin in which histidine 132 was replaced by an asparagine, were both unable to oligomerize and neither could form channels. This is evidence that oligomerization is a necessary step in channel formation.