Figure 6.
Figure 6. The rate of MMTS reaction with the native colicin E1 cysteine residue C505 in helix H9 depends on the charge of residue 473 in helix H8. (A) Wild-type colicin E1. Before the start of the record, wild-type colicin E1 was added to the cis compartment. Channels opened at +50 mV and remained open at −30 mV. The record begins with a step from 0 mV to +25 mV; the conductance increased slowly as more channels opened. After 4 min, at the arrows, 200 μg MMTS was added to both the trans and cis compartments. (The order of addition is not important.) This caused a large, slow decrease in conductance, which reached 83% in 40 min but was not yet complete. The decay was well fitted as a sum of two exponential components, representing decreases in conductance Af = 37% and As = 65%, with respective time constants τf = 173 s and τs = 1451 s. (Note that Af + As is the fitted total percentage conductance decrease, relative to the conductance before MTS addition, extrapolated to infinite time. Normally, this sum will be <100%, but in this panel it is slightly >100%, due to a small deviation of the fitted curve from the data.) (B) Colicin E1 mutant D473N. Before the start of the record, colicin E1 D473N was added to the cis compartment. As in A, channels opened at +50 mV and stayed open at −30 mV. (On average, the channel-forming activity of the mutant colicin, per nanogram added, is roughly 10 to 100 times lower than that of wild-type colicin E1; note the difference in current scale between A and B.) The record begins with a step from 0 to +30 mV. After a few minutes, at the arrows, 200 μg MMTS was added to both the cis and trans compartments. This produced a large decrease in conductance of 65% in 35 min, with clearly distinguished fast and slow components; the decay was adequately fitted as a sum of two exponential components, with amplitudes Af = 36% and As = 36%, and time constants τf = 15.5 s and τs = 797 s, respectively. In B, the sampled current points were not connected by lines to make the noise spikes less obtrusive. The solution on both sides of the membrane was as in Fig. 3. The amounts of colicin, octylglucoside, and DTT added to the cis compartment and DTT added to the trans compartment were, respectively, 585 ng, 4.5 μg, 5 μM, and 5 μM in A, and 338 ng, 4.5 μg, 10 μM, and 0 in B.

The rate of MMTS reaction with the native colicin E1 cysteine residue C505 in helix H9 depends on the charge of residue 473 in helix H8. (A) Wild-type colicin E1. Before the start of the record, wild-type colicin E1 was added to the cis compartment. Channels opened at +50 mV and remained open at −30 mV. The record begins with a step from 0 mV to +25 mV; the conductance increased slowly as more channels opened. After 4 min, at the arrows, 200 μg MMTS was added to both the trans and cis compartments. (The order of addition is not important.) This caused a large, slow decrease in conductance, which reached 83% in 40 min but was not yet complete. The decay was well fitted as a sum of two exponential components, representing decreases in conductance Af = 37% and As = 65%, with respective time constants τf = 173 s and τs = 1451 s. (Note that Af + As is the fitted total percentage conductance decrease, relative to the conductance before MTS addition, extrapolated to infinite time. Normally, this sum will be <100%, but in this panel it is slightly >100%, due to a small deviation of the fitted curve from the data.) (B) Colicin E1 mutant D473N. Before the start of the record, colicin E1 D473N was added to the cis compartment. As in A, channels opened at +50 mV and stayed open at −30 mV. (On average, the channel-forming activity of the mutant colicin, per nanogram added, is roughly 10 to 100 times lower than that of wild-type colicin E1; note the difference in current scale between A and B.) The record begins with a step from 0 to +30 mV. After a few minutes, at the arrows, 200 μg MMTS was added to both the cis and trans compartments. This produced a large decrease in conductance of 65% in 35 min, with clearly distinguished fast and slow components; the decay was adequately fitted as a sum of two exponential components, with amplitudes Af = 36% and As = 36%, and time constants τf = 15.5 s and τs = 797 s, respectively. In B, the sampled current points were not connected by lines to make the noise spikes less obtrusive. The solution on both sides of the membrane was as in Fig. 3. The amounts of colicin, octylglucoside, and DTT added to the cis compartment and DTT added to the trans compartment were, respectively, 585 ng, 4.5 μg, 5 μM, and 5 μM in A, and 338 ng, 4.5 μg, 10 μM, and 0 in B.

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