The paper by -hemolysin. The structure of the membrane-associated form of this protein has been solved to high resolution (
20 Å, and then widens into a large vestibule 46 Å across. The pore again narrows to
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 (
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 16, 19, 21, and 27 Å; all but the first too large to pass through the
-hemolysin pore. However, elongated configurations must be prevalent because all but the 5-kD reagent pass through the pore.
Cysteine-substituted -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 (
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
Bayley and co-workers (1 with cysteines on the near side of the constriction and much <1 with cysteines on the far side of the constriction.
Bayley and co-workers (
As expected from the discussion above, the rate constants of a given reagent with the different cysteines, in some cases with near neighbors, differed by orders of magnitude. The rate constants of the three larger reagents normalized by the rate constants for the 1-kD reagent, however, were more regular. With reagent on the cis side (as in Figure 1 of
The implications are different with the different reagents. The results with the 5-kD reagent indicated that there is a rate-limiting constriction between 106 and 113. The results with the 2.5-kD reagent indicated that there is a constriction between 106 and 117. The results with the 1.8-kD reagent did not bracket the constriction.
There is another way for us to normalize the rate constants: at each cysteine, we divide the rate constant for reagent added from the cis side by the rate constant for reagent added from the trans side. The basis for this normalization is the assumption that once a reagent molecule gets to a cysteine, the path by which it got there will not affect the local reaction rate (
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These ratios of rates reflect the steady-state reagent concentration at each cysteine after reagent was added to the cis side divided by the steady-state reagent concentration at the same cysteine after reagent was added to the trans side. For all four reagents, there is an obvious change in the ratios between S106 and K8. There was a significantly higher concentration of reagent at K8 for reagent added on the cis side than for reagent added on the trans side and vice versa for S106. After reagent is added to the cis side, the concentration at K8 is likely to be close to the bulk concentration in the medium on the cis side. After passing through the constriction between K8 and S106, however, the reagent is likely to be rapidly diluted in the large cavity; consequently, the steady-state reagent concentration is higher at K8 than at S106. Conversely, reagent coming from the trans side dilutes rapidly as it passes the constriction between S106 and K8 into the cis-side extramembranous medium; consequently the steady-state reagent concentration is higher at S106 than at K8. Thus, the ratios of rates are consistent with the minor constriction seen in the crystal structure.
With regard to the major constriction around M113, the ratios for the two larger reagents indicate a dividing line between significantly higher concentration from the cis side and significantly higher concentration from the trans side around M113. Thus, looked at two ways, the data of
Gates are channel obstructions that move, and SCAM has been used to locate gates in a voltage-gated K+ channel (
In the acetylcholine receptor, the region of the gate was accessible to small, charged methanethiosulfonate reagents from either side of the membrane, and it was possible to determine the ratio of rate constants both with respect to the side of application and with respect to the state of the gate (
SCAM requires some clues about the structure being probed. In the first instance, these clues guide the selection of residues to be mutated to cysteine. SCAM can provide rigorous tests of the assumed structures (
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Footnotes |
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1 The effect of steric hindrance along the pathway to a cysteine will depend on whether the concentration of reagent in the vicinity of the target cysteine is at equilibrium with the reagent in the medium or is in a steady state. When transfer rates are greater than the local reaction rate, reagent in a pore closed at one end, or reagent added to both sides of a pore open at both ends, will be at equilibrium with reagent in the medium. In contrast, reagent added to one side only of a pore that is open at both ends (the condition here) will be in a steady state. In the steady state, but not in the equilibrium state, the local concentration of reagent will be sensitive to steric hindrance along the pathway.
Submitted: 5 February 2001
Revised: 6 February 2001
Accepted: 6 February 2001
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References |
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