Limits...
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.

Show MeSH

Related in: MedlinePlus

The effect of trans trypsin on whole colicin Ia channels. Before the start of the record, 100 ng of whole colicin Ia were added to the cis compartment, and 5 μg of trypsin were added to the trans compartment. The record shows two channels opening at +40 mV. Each channel opened with the normal conductance (9 pS) for this salt condition, but then dropped to a low-conductance open state (0.9–1.0 pS). This decrease in conductance presumably reflects the cutting by trypsin of a site in the translocated segment and the subsequent separation of the helix 1 membrane-spanning segment from the rest of the channel. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM HEPES, pH 8.0. The record was filtered at 20 Hz.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230624&req=5

Figure 7: The effect of trans trypsin on whole colicin Ia channels. Before the start of the record, 100 ng of whole colicin Ia were added to the cis compartment, and 5 μg of trypsin were added to the trans compartment. The record shows two channels opening at +40 mV. Each channel opened with the normal conductance (9 pS) for this salt condition, but then dropped to a low-conductance open state (0.9–1.0 pS). This decrease in conductance presumably reflects the cutting by trypsin of a site in the translocated segment and the subsequent separation of the helix 1 membrane-spanning segment from the rest of the channel. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM HEPES, pH 8.0. The record was filtered at 20 Hz.

Mentions: As an independent approach to creating a colicin Ia fragment with only three membrane-spanning segments, we took advantage of the sensitivity of colicin Ia to protease in the trans solution (Kagan 1981). After adding whole colicin Ia to the cis compartment, we attempted to cut off the fourth, amino-terminal–most segment of the open channel (along with the T and R domains) using trypsin in the trans compartment. Based on our model of the whole colicin Ia open channel (Fig. 1), the only trypsin sites exposed on the trans side of the membrane should be the 15 or 16 basic residues in the translocated segment, from K470 or R476 to R537. As shown in Fig. 7, trans trypsin caused the conductance of single channels to drop from the normal conductance (in 100 mM KCl, pH 8.0) of 9 pS to a low-conductance state of 1 pS, in a manner reminiscent of the spontaneous behavior of C domain channels. This effect was not observed in the absence of trypsin and could be prevented by the addition of soybean trypsin inhibitor to the trans solution, demonstrating that it is a specific enzymatic effect. In 1 M KCl, pH 8.0, trans trypsin made the conductance drop from the normal conductance (at this pH and salt concentration) of 60 pS down to 8 pS. Unpublished experiments with a whole colicin Ia mutant indicate that the major trypsin site is the pair of lysine residues (485–486) in the loop between C domain helices 2 and 3.


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 trypsin on whole colicin Ia channels. Before the start of the record, 100 ng of whole colicin Ia were added to the cis compartment, and 5 μg of trypsin were added to the trans compartment. The record shows two channels opening at +40 mV. Each channel opened with the normal conductance (9 pS) for this salt condition, but then dropped to a low-conductance open state (0.9–1.0 pS). This decrease in conductance presumably reflects the cutting by trypsin of a site in the translocated segment and the subsequent separation of the helix 1 membrane-spanning segment from the rest of the channel. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM HEPES, pH 8.0. The record was filtered at 20 Hz.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: The effect of trans trypsin on whole colicin Ia channels. Before the start of the record, 100 ng of whole colicin Ia were added to the cis compartment, and 5 μg of trypsin were added to the trans compartment. The record shows two channels opening at +40 mV. Each channel opened with the normal conductance (9 pS) for this salt condition, but then dropped to a low-conductance open state (0.9–1.0 pS). This decrease in conductance presumably reflects the cutting by trypsin of a site in the translocated segment and the subsequent separation of the helix 1 membrane-spanning segment from the rest of the channel. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM HEPES, pH 8.0. The record was filtered at 20 Hz.
Mentions: As an independent approach to creating a colicin Ia fragment with only three membrane-spanning segments, we took advantage of the sensitivity of colicin Ia to protease in the trans solution (Kagan 1981). After adding whole colicin Ia to the cis compartment, we attempted to cut off the fourth, amino-terminal–most segment of the open channel (along with the T and R domains) using trypsin in the trans compartment. Based on our model of the whole colicin Ia open channel (Fig. 1), the only trypsin sites exposed on the trans side of the membrane should be the 15 or 16 basic residues in the translocated segment, from K470 or R476 to R537. As shown in Fig. 7, trans trypsin caused the conductance of single channels to drop from the normal conductance (in 100 mM KCl, pH 8.0) of 9 pS to a low-conductance state of 1 pS, in a manner reminiscent of the spontaneous behavior of C domain channels. This effect was not observed in the absence of trypsin and could be prevented by the addition of soybean trypsin inhibitor to the trans solution, demonstrating that it is a specific enzymatic effect. In 1 M KCl, pH 8.0, trans trypsin made the conductance drop from the normal conductance (at this pH and salt concentration) of 60 pS down to 8 pS. Unpublished experiments with a whole colicin Ia mutant indicate that the major trypsin site is the pair of lysine residues (485–486) in the loop between C domain helices 2 and 3.

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