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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 a longer carboxy-terminal fragment with a biotin attached near the amino terminus. Before the start of the record, 240 ng of biotinylated mutant 326C/CT-L (with an amino-terminal His-tag) were added to the cis compartment. The conductance turned on at +70 mV and off at −80 mV. At the arrow, 20 μg of streptavidin were added to the trans compartment. This produced an inhibition of turn off at negative voltage, with the development of a noisy conductance. This shows that the amino-terminal biotin of this long fragment 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 HEPES, pH 7.2. The record was filtered at 30 Hz.
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Figure 4: The effect of trans streptavidin on a longer carboxy-terminal fragment with a biotin attached near the amino terminus. Before the start of the record, 240 ng of biotinylated mutant 326C/CT-L (with an amino-terminal His-tag) were added to the cis compartment. The conductance turned on at +70 mV and off at −80 mV. At the arrow, 20 μg of streptavidin were added to the trans compartment. This produced an inhibition of turn off at negative voltage, with the development of a noisy conductance. This shows that the amino-terminal biotin of this long fragment 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 HEPES, pH 7.2. The record was filtered at 30 Hz.

Mentions: To determine whether a sequence upstream from the C domain acts as an anchor to stop translocation, we prepared a much longer carboxy-terminal fragment of colicin Ia (CT-L), including all of the long helix 1 extending from the C domain, plus part of the R domain, and attached biotin near its amino terminus at residue 326. This mutant also responded to trans streptavidin; the effect was an inhibition of turn off at negative voltage (more like the effect that we had originally anticipated for the shorter fragments), with the development of a noisy conductance (Fig. 4). Hence, the amino terminus of this long fragment moves across the membrane to the trans side. We obtained a similar result when the biotin was attached at residue 381, ∼80 residues from the amino terminus of this fragment (including 25 residues from the His-tag sequence) (data not shown), suggesting that not only the amino terminus, but probably all of the amino-terminal region, is translocated. The effect of trans streptavidin on both mutants was prevented by the earlier addition of excess biotin to the trans solution.


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 a longer carboxy-terminal fragment with a biotin attached near the amino terminus. Before the start of the record, 240 ng of biotinylated mutant 326C/CT-L (with an amino-terminal His-tag) were added to the cis compartment. The conductance turned on at +70 mV and off at −80 mV. At the arrow, 20 μg of streptavidin were added to the trans compartment. This produced an inhibition of turn off at negative voltage, with the development of a noisy conductance. This shows that the amino-terminal biotin of this long fragment 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 HEPES, pH 7.2. The record was filtered at 30 Hz.
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Related In: Results  -  Collection

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Figure 4: The effect of trans streptavidin on a longer carboxy-terminal fragment with a biotin attached near the amino terminus. Before the start of the record, 240 ng of biotinylated mutant 326C/CT-L (with an amino-terminal His-tag) were added to the cis compartment. The conductance turned on at +70 mV and off at −80 mV. At the arrow, 20 μg of streptavidin were added to the trans compartment. This produced an inhibition of turn off at negative voltage, with the development of a noisy conductance. This shows that the amino-terminal biotin of this long fragment 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 HEPES, pH 7.2. The record was filtered at 30 Hz.
Mentions: To determine whether a sequence upstream from the C domain acts as an anchor to stop translocation, we prepared a much longer carboxy-terminal fragment of colicin Ia (CT-L), including all of the long helix 1 extending from the C domain, plus part of the R domain, and attached biotin near its amino terminus at residue 326. This mutant also responded to trans streptavidin; the effect was an inhibition of turn off at negative voltage (more like the effect that we had originally anticipated for the shorter fragments), with the development of a noisy conductance (Fig. 4). Hence, the amino terminus of this long fragment moves across the membrane to the trans side. We obtained a similar result when the biotin was attached at residue 381, ∼80 residues from the amino terminus of this fragment (including 25 residues from the His-tag sequence) (data not shown), suggesting that not only the amino terminus, but probably all of the amino-terminal region, is translocated. The effect of trans streptavidin on both mutants was prevented by the earlier addition of excess biotin to the trans solution.

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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