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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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Comparison of single channels of whole colicin Ia with its carboxy-terminal fragments. (A) Two whole colicin Ia channels opened with a relatively high conductance (42–44 pS) and stayed at this level. (B) Four C domain channels in succession opened with a conductance comparable with whole colicin channels (33–44 pS), but then dropped to a state of lower conductance (5–7 pS). (C) Example of one C domain channel that passed back through the higher-conductance state (here 33 pS) before closing at negative voltage. (D) Schematic model of whole colicin Ia in its open state, with four membrane-spanning segments. (E) Model of C domain in the transient, higher-conductance state, also with four membrane-spanning segments. (F) Model of C domain in the low-conductance open state, with only three membrane-spanning segments, and helix H1 translocated across the membrane. The segments labeled H1 in D–F represent the part of helix 1 that is within the C domain. The white bars in A indicate the open channel state of whole colicin Ia that is diagrammed in D. In B and C, the black bars indicate when the channel is in the transient open state of E, and the gray bars indicate when it is in the low-conductance open state of F. The amount of colicin added to the cis compartment was: (A) 3 ng whole colicin Ia, (B) 0.37 ng CT-M without a His-tag, and (C) 10 ng biotinylated mutant 453C/CT-S with an amino-terminal His-tag. The records were filtered at (A) 30 Hz, (B) 20 Hz, (C) 10 Hz, and (D) none of the above. The solution on both sides of the membrane for A–C was 1 M KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM MES, pH 6.2. B begins with four channels already open in the low-conductance state; there are no channels open at the beginning of A or C.
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Figure 5: Comparison of single channels of whole colicin Ia with its carboxy-terminal fragments. (A) Two whole colicin Ia channels opened with a relatively high conductance (42–44 pS) and stayed at this level. (B) Four C domain channels in succession opened with a conductance comparable with whole colicin channels (33–44 pS), but then dropped to a state of lower conductance (5–7 pS). (C) Example of one C domain channel that passed back through the higher-conductance state (here 33 pS) before closing at negative voltage. (D) Schematic model of whole colicin Ia in its open state, with four membrane-spanning segments. (E) Model of C domain in the transient, higher-conductance state, also with four membrane-spanning segments. (F) Model of C domain in the low-conductance open state, with only three membrane-spanning segments, and helix H1 translocated across the membrane. The segments labeled H1 in D–F represent the part of helix 1 that is within the C domain. The white bars in A indicate the open channel state of whole colicin Ia that is diagrammed in D. In B and C, the black bars indicate when the channel is in the transient open state of E, and the gray bars indicate when it is in the low-conductance open state of F. The amount of colicin added to the cis compartment was: (A) 3 ng whole colicin Ia, (B) 0.37 ng CT-M without a His-tag, and (C) 10 ng biotinylated mutant 453C/CT-S with an amino-terminal His-tag. The records were filtered at (A) 30 Hz, (B) 20 Hz, (C) 10 Hz, and (D) none of the above. The solution on both sides of the membrane for A–C was 1 M KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM MES, pH 6.2. B begins with four channels already open in the low-conductance state; there are no channels open at the beginning of A or C.

Mentions: The conductance of whole colicin Ia channels is 40–44 pS in 1 M KCl, pH 6.2 (Fig. 5 A). At a voltage of +50 mV or more, channels remain in this conductance state “indefinitely” (that is, the patience of the channel is greater than that of the experimenter), with occasional brief flickers to a fully closed state. C domain channels show a different behavior. These channels open initially with about the same conductance (33–44 pS) as that of whole colicin Ia channels, but then quickly pass to a state of lower conductance (5–10 pS) (Fig. 5 B). This low-conductance open state is stable: channels do not return to the normal-conductance state while the positive voltage is maintained. Our interpretation of these results is that we have resolved the transition of the C domain portion of helix 1 from a membrane-spanning conformation to a completely translocated state. According to this view, the transient higher-conductance state corresponds to a channel with four membrane-spanning segments, resembling the whole colicin Ia channel (Fig. 5 E), and the low-conductance state comes from a channel with only three membrane-spanning segments (F). Qualitatively similar results were obtained with all the carboxy-terminal fragments discussed so far (CT-S, CT-M, and CT-L) and did not depend on the presence of an amino-terminal His-tag or biotin. Sometimes, the channels passed back through the higher-conductance state before closing at negative voltage (Fig. 5 C). This suggests that en route to closing, the amino terminus of the C domain can reinsert into the membrane from the trans side, while the downstream membrane-spanning segments are still in place.


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)

Comparison of single channels of whole colicin Ia with its carboxy-terminal fragments. (A) Two whole colicin Ia channels opened with a relatively high conductance (42–44 pS) and stayed at this level. (B) Four C domain channels in succession opened with a conductance comparable with whole colicin channels (33–44 pS), but then dropped to a state of lower conductance (5–7 pS). (C) Example of one C domain channel that passed back through the higher-conductance state (here 33 pS) before closing at negative voltage. (D) Schematic model of whole colicin Ia in its open state, with four membrane-spanning segments. (E) Model of C domain in the transient, higher-conductance state, also with four membrane-spanning segments. (F) Model of C domain in the low-conductance open state, with only three membrane-spanning segments, and helix H1 translocated across the membrane. The segments labeled H1 in D–F represent the part of helix 1 that is within the C domain. The white bars in A indicate the open channel state of whole colicin Ia that is diagrammed in D. In B and C, the black bars indicate when the channel is in the transient open state of E, and the gray bars indicate when it is in the low-conductance open state of F. The amount of colicin added to the cis compartment was: (A) 3 ng whole colicin Ia, (B) 0.37 ng CT-M without a His-tag, and (C) 10 ng biotinylated mutant 453C/CT-S with an amino-terminal His-tag. The records were filtered at (A) 30 Hz, (B) 20 Hz, (C) 10 Hz, and (D) none of the above. The solution on both sides of the membrane for A–C was 1 M KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM MES, pH 6.2. B begins with four channels already open in the low-conductance state; there are no channels open at the beginning of A or C.
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Figure 5: Comparison of single channels of whole colicin Ia with its carboxy-terminal fragments. (A) Two whole colicin Ia channels opened with a relatively high conductance (42–44 pS) and stayed at this level. (B) Four C domain channels in succession opened with a conductance comparable with whole colicin channels (33–44 pS), but then dropped to a state of lower conductance (5–7 pS). (C) Example of one C domain channel that passed back through the higher-conductance state (here 33 pS) before closing at negative voltage. (D) Schematic model of whole colicin Ia in its open state, with four membrane-spanning segments. (E) Model of C domain in the transient, higher-conductance state, also with four membrane-spanning segments. (F) Model of C domain in the low-conductance open state, with only three membrane-spanning segments, and helix H1 translocated across the membrane. The segments labeled H1 in D–F represent the part of helix 1 that is within the C domain. The white bars in A indicate the open channel state of whole colicin Ia that is diagrammed in D. In B and C, the black bars indicate when the channel is in the transient open state of E, and the gray bars indicate when it is in the low-conductance open state of F. The amount of colicin added to the cis compartment was: (A) 3 ng whole colicin Ia, (B) 0.37 ng CT-M without a His-tag, and (C) 10 ng biotinylated mutant 453C/CT-S with an amino-terminal His-tag. The records were filtered at (A) 30 Hz, (B) 20 Hz, (C) 10 Hz, and (D) none of the above. The solution on both sides of the membrane for A–C was 1 M KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM MES, pH 6.2. B begins with four channels already open in the low-conductance state; there are no channels open at the beginning of A or C.
Mentions: The conductance of whole colicin Ia channels is 40–44 pS in 1 M KCl, pH 6.2 (Fig. 5 A). At a voltage of +50 mV or more, channels remain in this conductance state “indefinitely” (that is, the patience of the channel is greater than that of the experimenter), with occasional brief flickers to a fully closed state. C domain channels show a different behavior. These channels open initially with about the same conductance (33–44 pS) as that of whole colicin Ia channels, but then quickly pass to a state of lower conductance (5–10 pS) (Fig. 5 B). This low-conductance open state is stable: channels do not return to the normal-conductance state while the positive voltage is maintained. Our interpretation of these results is that we have resolved the transition of the C domain portion of helix 1 from a membrane-spanning conformation to a completely translocated state. According to this view, the transient higher-conductance state corresponds to a channel with four membrane-spanning segments, resembling the whole colicin Ia channel (Fig. 5 E), and the low-conductance state comes from a channel with only three membrane-spanning segments (F). Qualitatively similar results were obtained with all the carboxy-terminal fragments discussed so far (CT-S, CT-M, and CT-L) and did not depend on the presence of an amino-terminal His-tag or biotin. Sometimes, the channels passed back through the higher-conductance state before closing at negative voltage (Fig. 5 C). This suggests that en route to closing, the amino terminus of the C domain can reinsert into the membrane from the trans side, while the downstream membrane-spanning segments are still in place.

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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