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Protein translocation across planar bilayers by the colicin Ia channel-forming domain: where will it end?

Kienker PK, Jakes KS, Finkelstein A - J. Gen. Physiol. (2000)

Bottom Line: To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand.The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane.Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. kienker@aecom.yu.edu

ABSTRACT
Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an approximately 68-residue segment translocated across the membrane. Various experimental interventions could cause a longer or shorter segment within the C domain to be translocated, making us wonder why translocation normally stops where it does, near the amino-terminal end of the C domain (approximately residue 450). We hypothesized that regions upstream from the C domain prevent its amino-terminal end from moving into and across the membrane. To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand. The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane. Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane. All of the colicin Ia carboxy-terminal fragments that we examined form channels that pass from a state of relatively normal conductance to a low-conductance state; we interpret this passage as a transition from a channel with four membrane-spanning segments to one with only three.

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The effect of trans streptavidin on C domain with biotin attached near its amino terminus. Before the start of the record, 20 ng of biotinylated mutant 453C/CT-S without a His-tag (plus 1 μg of octyl glucoside) were added to the cis compartment. Normal gating was seen, with the conductance turning on at +70 and off at −70 mV. At the arrow, 20 μg of streptavidin was added to the trans compartment. A new conductance rapidly developed that turned on at −70 and off at 0 or +70 mV—the reverse of the normal voltage dependence. (The turn off of the “reversed” conductance during a pulse to 0 or +70 mV was evident from the decreased conductance at −70 mV just after the pulse, relative to that just before the pulse.) After streptavidin addition, there were two populations of channel in the membrane: the original population of normal-gating channels that had not yet bound trans streptavidin, and a new population of reverse-gating channels that had. This shows that the amino-terminal biotin was accessible to trans streptavidin. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM MES, pH 6.2. The record was filtered at 30 Hz.
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Figure 3: The effect of trans streptavidin on C domain with biotin attached near its amino terminus. Before the start of the record, 20 ng of biotinylated mutant 453C/CT-S without a His-tag (plus 1 μg of octyl glucoside) were added to the cis compartment. Normal gating was seen, with the conductance turning on at +70 and off at −70 mV. At the arrow, 20 μg of streptavidin was added to the trans compartment. A new conductance rapidly developed that turned on at −70 and off at 0 or +70 mV—the reverse of the normal voltage dependence. (The turn off of the “reversed” conductance during a pulse to 0 or +70 mV was evident from the decreased conductance at −70 mV just after the pulse, relative to that just before the pulse.) After streptavidin addition, there were two populations of channel in the membrane: the original population of normal-gating channels that had not yet bound trans streptavidin, and a new population of reverse-gating channels that had. This shows that the amino-terminal biotin was accessible to trans streptavidin. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM MES, pH 6.2. The record was filtered at 30 Hz.

Mentions: Fig. 3 shows a typical experiment. Biotinylated C domain was added to the cis solution and the normal pattern of gating was established. C domain channel gating is similar to that of whole colicin Ia channels, with turn-on at positive voltage and turn-off at negative voltage; however, turn-off is slower for C domain channels than for whole colicin Ia channels. After streptavidin was added to the trans solution, a new sort of conductance quickly appeared that turned on at negative voltage and off at zero or positive voltage–the reverse of the normal voltage dependence. This was not the anticipated effect of trans streptavidin, but it nevertheless shows that the amino-terminal biotin crossed the membrane to the trans side. The effect was the same for either C-domain fragment, CT-S or CT-M; it was also the same with or without the amino-terminal His-tag. The effect of trans streptavidin on biotinylated CT-S and CT-M was prevented by the earlier addition of excess biotin to the trans solution. Streptavidin had no effect on unbiotinylated C-domain fragments.


Protein translocation across planar bilayers by the colicin Ia channel-forming domain: where will it end?

Kienker PK, Jakes KS, Finkelstein A - J. Gen. Physiol. (2000)

The effect of trans streptavidin on C domain with biotin attached near its amino terminus. Before the start of the record, 20 ng of biotinylated mutant 453C/CT-S without a His-tag (plus 1 μg of octyl glucoside) were added to the cis compartment. Normal gating was seen, with the conductance turning on at +70 and off at −70 mV. At the arrow, 20 μg of streptavidin was added to the trans compartment. A new conductance rapidly developed that turned on at −70 and off at 0 or +70 mV—the reverse of the normal voltage dependence. (The turn off of the “reversed” conductance during a pulse to 0 or +70 mV was evident from the decreased conductance at −70 mV just after the pulse, relative to that just before the pulse.) After streptavidin addition, there were two populations of channel in the membrane: the original population of normal-gating channels that had not yet bound trans streptavidin, and a new population of reverse-gating channels that had. This shows that the amino-terminal biotin was accessible to trans streptavidin. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM MES, pH 6.2. The record was filtered at 30 Hz.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: The effect of trans streptavidin on C domain with biotin attached near its amino terminus. Before the start of the record, 20 ng of biotinylated mutant 453C/CT-S without a His-tag (plus 1 μg of octyl glucoside) were added to the cis compartment. Normal gating was seen, with the conductance turning on at +70 and off at −70 mV. At the arrow, 20 μg of streptavidin was added to the trans compartment. A new conductance rapidly developed that turned on at −70 and off at 0 or +70 mV—the reverse of the normal voltage dependence. (The turn off of the “reversed” conductance during a pulse to 0 or +70 mV was evident from the decreased conductance at −70 mV just after the pulse, relative to that just before the pulse.) After streptavidin addition, there were two populations of channel in the membrane: the original population of normal-gating channels that had not yet bound trans streptavidin, and a new population of reverse-gating channels that had. This shows that the amino-terminal biotin was accessible to trans streptavidin. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM MES, pH 6.2. The record was filtered at 30 Hz.
Mentions: Fig. 3 shows a typical experiment. Biotinylated C domain was added to the cis solution and the normal pattern of gating was established. C domain channel gating is similar to that of whole colicin Ia channels, with turn-on at positive voltage and turn-off at negative voltage; however, turn-off is slower for C domain channels than for whole colicin Ia channels. After streptavidin was added to the trans solution, a new sort of conductance quickly appeared that turned on at negative voltage and off at zero or positive voltage–the reverse of the normal voltage dependence. This was not the anticipated effect of trans streptavidin, but it nevertheless shows that the amino-terminal biotin crossed the membrane to the trans side. The effect was the same for either C-domain fragment, CT-S or CT-M; it was also the same with or without the amino-terminal His-tag. The effect of trans streptavidin on biotinylated CT-S and CT-M was prevented by the earlier addition of excess biotin to the trans solution. Streptavidin had no effect on unbiotinylated C-domain fragments.

Bottom Line: To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand.The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane.Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. kienker@aecom.yu.edu

ABSTRACT
Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an approximately 68-residue segment translocated across the membrane. Various experimental interventions could cause a longer or shorter segment within the C domain to be translocated, making us wonder why translocation normally stops where it does, near the amino-terminal end of the C domain (approximately residue 450). We hypothesized that regions upstream from the C domain prevent its amino-terminal end from moving into and across the membrane. To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand. The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane. Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane. All of the colicin Ia carboxy-terminal fragments that we examined form channels that pass from a state of relatively normal conductance to a low-conductance state; we interpret this passage as a transition from a channel with four membrane-spanning segments to one with only three.

Show MeSH
Related in: MedlinePlus