Scam Feels the Pinch

The paper by Movileanu et al. (2001) in this issue of The Journal describes a test of the applicability of the substituted-cysteine-accessibility method (SCAM; Akabas et al., 1992; Akabas et al., 1994) to the problem of locating the narrowest region of a channel lumen. Movileanu et al. (2001) use unusually large sulfhydryldirected reagents to probe the wide bore channel formed by Staphylococcal a -hemolysin. The structure of the membrane-associated form of this protein has been solved to high resolution (Song et al., 1996). It is a mushroom-shaped heptameric complex that inserts in the membrane of a susceptible cell with the wide cap on the extracellular side, or on the cis side of an artificial planar lipid membrane, and with the stem traversing the bilayer (see Figure 1 in Movileanu et al., 2001). The entrance to the pore at the extracellular or cis end is 30 Å wide. The pore immediately narrows to z 20 Å, and then widens into a large vestibule 46 Å across. The pore again narrows to z 16 Å, its major constriction, and then continues to its intracellular or trans end as an irregular cylinder, which is roughly 20 Å wide. Bayley and co-workers (Movileanu et al., 2001) demonstrate that they can locate the narrowest constriction of the pore by applying SCAM with large enough reagents. The reagents were 2-pyridyl disulfide derivatives of polyethylene glycol (PEG) of average molecular masses: 1,000, 1,800, 2,500 and 5,000 D. They react to form mixed disulfides with cysteine in which the PEG moiety is attached to the cysteine sulfur. If these reagents were unhydrated spheres, their diameters would be z 16, 19, 21, and 27 Å; all but the first too large to pass through the a -hemolysin pore. However, elongated configurations must be prevalent because all but the 5-kD reagent pass through the pore. Cysteine-substituted a -hemolysin mutants were synthesized in vitro by coupled transcription and translation, purified, and incorporated into artificial planar bilayers. The reactions of the cysteines with the PEG reagents were monitored by their effects on the conductance of the pore, which were irreversible by washing but reversible by reduction. The basis for locating the narrowest constriction of the channel is simply that, all other things being equal, the rate of reaction of a reagent added to one side of the membrane with a cysteine on the near side of the constriction will be faster than the rate of reaction with a cysteine on the far side of the constriction. However, all other things are seldom equal. Numerous factors can influence the reaction rate of a cysteine in a pore (Wilson and Karlin, 2001). There are two kinds of processes: (1) the transfer of reagent to and from the vicinity of the cysteine, which determines the local concentration of the reagent; and (2) the reaction of local reagent with the cysteine (Pascual and Karlin, 1998b). The transfer rate constants depend on steric hindrance and electrostatics along the pathway to the cysteine, whereas the local reaction rate constant depends on local steric hindrance and electrostatics. The reaction considered here with mercaptopyridine derivatives, like the reaction with methanethiosulfonate derivatives, takes place exclusively with the deprotonated cysteine sulfhydryl (Roberts et al., 1986). Hence, the local reaction rate depends on the sulfhydryl pK a and the local pH, which also depends on local electrostatic interactions. All of the factors influencing the overall reaction rate are likely to be different at different locations in the pore. How then can reaction rates be used to locate a constriction in the pore? The trick is to normalize the rates so that all factors cancel except for the effect of the constriction. The reagents, of course, must be large enough that their transfer rate constants are significantly lowered by the constriction, and a large enough number of substituted cysteines must be tested to bracket the constriction and to establish a pattern of reactivity on either side of it. 1