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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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Schematic diagram of the whole colicin Ia molecule in the open channel state in a planar bilayer. Helices 1–10 of the carboxy-terminal C domain are labeled to show their topology in the membrane, with four membrane-spanning segments (helices 8 and 9 and portions of helices 1 and 6–7) and a translocated segment (helices 2–5). The C domain begins at about residue 451, in helix 1, which is located near the cis interface. The amino-terminal T domain and the central R domain, as well as the two long inter-domain helices, are presumed to reside on the cis side of the membrane.
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Figure 1: Schematic diagram of the whole colicin Ia molecule in the open channel state in a planar bilayer. Helices 1–10 of the carboxy-terminal C domain are labeled to show their topology in the membrane, with four membrane-spanning segments (helices 8 and 9 and portions of helices 1 and 6–7) and a translocated segment (helices 2–5). The C domain begins at about residue 451, in helix 1, which is located near the cis interface. The amino-terminal T domain and the central R domain, as well as the two long inter-domain helices, are presumed to reside on the cis side of the membrane.

Mentions: Colicin Ia belongs to a family of water-soluble bactericidal proteins that consist of three domains: the central R domain and the amino-terminal T domain are responsible for receptor-binding and translocation of the colicin across the outer membrane of the target cell, respectively, and the carboxy-terminal C domain forms a channel in the inner membrane to kill the cell (for general review, see Cramer et al. 1995). The crystal structure of the water-soluble form of colicin Ia reveals that the three domains are separated by two long α-helices in a coiled-coil, making a “Y”-shaped molecule (Wiener et al. 1997). Aside from the hydrophobic hairpin formed by helices 8 and 9 of the C domain, the rest of the molecule is highly charged (>30% of the residues) (Mankovich et al. 1986). Whole colicin or isolated C domain can also form channels in planar lipid bilayer membranes (Nogueira and Varanda 1988; Ghosh et al. 1993). When whole colicin Ia associates with a planar bilayer, it undergoes a series of conformational changes: after the hydrophobic hairpin inserts into the membrane (Kienker et al. 1997), the conducting channel is formed by the voltage-dependent insertion of two additional segments, with portions of helix 1 and helices 6–7 spanning the membrane, and helices 2–5 translocated completely across the membrane (Qiu et al. 1996) (Fig. 1). The T and R domains presumably remain on the cis side (the side to which the colicin was added).


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)

Schematic diagram of the whole colicin Ia molecule in the open channel state in a planar bilayer. Helices 1–10 of the carboxy-terminal C domain are labeled to show their topology in the membrane, with four membrane-spanning segments (helices 8 and 9 and portions of helices 1 and 6–7) and a translocated segment (helices 2–5). The C domain begins at about residue 451, in helix 1, which is located near the cis interface. The amino-terminal T domain and the central R domain, as well as the two long inter-domain helices, are presumed to reside on the cis side of the membrane.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230624&req=5

Figure 1: Schematic diagram of the whole colicin Ia molecule in the open channel state in a planar bilayer. Helices 1–10 of the carboxy-terminal C domain are labeled to show their topology in the membrane, with four membrane-spanning segments (helices 8 and 9 and portions of helices 1 and 6–7) and a translocated segment (helices 2–5). The C domain begins at about residue 451, in helix 1, which is located near the cis interface. The amino-terminal T domain and the central R domain, as well as the two long inter-domain helices, are presumed to reside on the cis side of the membrane.
Mentions: Colicin Ia belongs to a family of water-soluble bactericidal proteins that consist of three domains: the central R domain and the amino-terminal T domain are responsible for receptor-binding and translocation of the colicin across the outer membrane of the target cell, respectively, and the carboxy-terminal C domain forms a channel in the inner membrane to kill the cell (for general review, see Cramer et al. 1995). The crystal structure of the water-soluble form of colicin Ia reveals that the three domains are separated by two long α-helices in a coiled-coil, making a “Y”-shaped molecule (Wiener et al. 1997). Aside from the hydrophobic hairpin formed by helices 8 and 9 of the C domain, the rest of the molecule is highly charged (>30% of the residues) (Mankovich et al. 1986). Whole colicin or isolated C domain can also form channels in planar lipid bilayer membranes (Nogueira and Varanda 1988; Ghosh et al. 1993). When whole colicin Ia associates with a planar bilayer, it undergoes a series of conformational changes: after the hydrophobic hairpin inserts into the membrane (Kienker et al. 1997), the conducting channel is formed by the voltage-dependent insertion of two additional segments, with portions of helix 1 and helices 6–7 spanning the membrane, and helices 2–5 translocated completely across the membrane (Qiu et al. 1996) (Fig. 1). The T and R domains presumably remain on the cis side (the side to which the colicin was added).

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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