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The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, [([PA.sub.63]).sub.7], formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that [([PA.sub.63]).sub.7] incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although [LF.sub.N] (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the [([PA.sub.63]).sub.7] channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on [LF.sub.N] enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton-protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one S[O.sub.3.sup.-], which is essentially not titratable, is introduced at most positions in [LF.sub.N], through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethio-sulfonate, voltage-driven [LF.sub.N] translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the [([PA.sub.63]).sub.7] channel resides at or near its [PHI]-clamp, the ring of seven phenylalanines near the channel's entrance.

Colicin Ia is a bactericidal protein of 626 amino acid residues that kills its target cell by forming a channel in the inner membrane; it can also form voltage-dependent channels in planar lipid bilayer membranes. The channel-forming activity resides in the carboxy-terminal domain of ~177 residues. In the crystal structure of the water-soluble conformation, this domain consists of a bundle of 10 [alpha]-helices, with eight mostly amphipathic helices surrounding a hydrophobic helical hairpin (helices H8-H9). We wish to know how this structure changes to form a channel in a lipid bilayer. Although there is evidence that the open channel has four transmembrane segments (H8, H9, and parts of H1 and H6-H7), their arrangement relative to the pore is largely unknown. Given the lack of a detailed structural model, it is imperative to better characterize the channel-lining protein segments. Here, we focus on a segment of 44 residues (573-616), which in the crystal structure comprises the H8-H9 hairpin and flanking regions. We mutated each of these residues to a unique cysteine, added the mutant colicins to the cis side of planar bilayers to form channels, and determined whether sulfhydryl-specific methanethiosulfonate reagents could alter the conduction of ions through the open channel. We found a pattern of reactivity consistent with parts of H8 and H9 lining the channel as [alpha]-helices, albeit rather short ones for spanning a lipid bilayer (12 residues). The effects of the reactions on channel conductance and selectivity tend to be greater for residues near the amino terminus of H8 and the carboxy terminus of H9, with particularly large effects for G577C, T581C, and G609C, suggesting that these residues may occupy a relatively constricted region near the cis end of the channel.

The bacterial toxin colicin Ia forms voltage-gated channels in planar lipid bilayers. The toxin consists of three domains, with the carboxy-terminal domain (C-domain) responsible for channel formation. The C-domain contributes four membrane-spanning segments and a 68-residue translocated segment to the open channel, whereas the upstream domains and the amino-terminal end of the C-domain stay on the cis side of the membrane. The isolated C-domain, lacking the two upstream domains, also forms channels; however, the amino terminus and one of the normally membrane-spanning segments can move across the membrane. (This can be observed as a drop in single-channel conductance.) In longer carboxy-terminal fragments of colicin Ia that include [less than or equal to] 169 residues upstream from the C-domain, the entire upstream region is translocated. Presumably, a portion of the C-domain creates a pathway for the polar upstream region to move through the membrane. To determine the size of this translocation pathway, we have attached 'molecular stoppers,' small disulfide-bonded polypeptides, to the amino terminus of the C-domain, and determined whether they could be translocated. We have found that the translocation rate is strongly voltage dependent, and that at voltages [greater than or equal to] 90 mV, even a 26-[Angstrom] stopper is translocated. Upon reduction of their disulfide bonds, all of the stoppers are easily translocated, indicating that it is the folded structure, rather than some aspect of the primary sequence, that slows translocation of the stoppers. Thus, the pathway for translocation is [greater than or equal to] 26 [Angstrom] in diameter, or can stretch to this value. This is large enough for an [alpha]-helical hairpin to fit through. KEY WORDS: [alpha]-conotoxin * apamin * charybdotoxin * disulfides * molecular stoppers

Research has been conducted on the anthrax toxin which consists of the projective antigen (PA), lethal factor and edema factor. Results indicate that PA (sub)63 is responsible for the toxin's catalytic fragment delivery to the target cell cytosol.

In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as [glucosamine.sup.+] and [NAD.sup.-], it would appear that the channel must be a multimer. Yet, there is substantial circumstantial evidence that the channel may be formed from a single subunit. To test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric, we made mixtures of two T domain constructs whose voltage-gating characteristics differ, and then observed the gating behavior of the mixture's single channels in planar lipid bilayers. One of these constructs contained an [NH.sub.2]-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a COOH-terminal H6 tag that blocks the channel at positive voltages. If the channel is constructed from multiple T domain subunits, one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. Therefore, we conclude that the T domain channel is monomeric. KEY WORDS: planar lipid bilayers * histidine tags * voltage gating * single channels * monomer

A discussion of Nicholas Barbon's economic theories is presented, focusing on seventeenth-century balance of trade theory. Topics include money, value, interest, wealth, infinite demand, and infinite supply.

The diphtheria toxin T domain, extending from residue 202 to 378, leads to translocation of the catalytic A fragment over endosomal membranes, and forms ion-conducting channels in planar phospholipid bilayers. The NH(s8b2) terminal region of the T domain has been investigated. Normal channel formation by the T domain was found to require that its NH(sub2) terminus be translocated over the membrane from the cis to the trans side.

A study was conducted to identify the residues lining the anthrax protective antigen channel. The results reveal alternating reduction and absence of reduction of conductance in consecutive residues over two stretches. The findings provide support for the model of conversion of the prepore to a 14-stranded Beta barrel pore and fortify the foundation for future research on the mechanism of translocation by anthrax toxin.

It has been established that the 61 amino acid stretch from residue 322 to 382 in the T-domain of diphtheria toxin forms channels indistinguishable from those in the whole T-domain. A study was undertaken to determine which residues in TH8-9 line the ion-conducting pathway of the channel. It was deduced that the channel is not multimeric is is formed from just on TH8-9 unit of the T-domain.

Colicin Ia forms channels in phospholipid bilayers as directed by voltage changes. The parts of the membrane involved during the opening and closing of channels were identified with the addition of streptavidin. Two transmembrane segments, upstream and downstream, were found to form in association with channel gating. The addition of streptavidin to the cis phase prevented channel opening but had no effect on the other opened channels thereby allowing a segment of the translocated protein to be a part of the upstream segment inserted into the bilayer.