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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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C domain with its amino terminus anchored on the cis side by streptavidin made normal channels. A preincubated mixture containing 4 ng of biotinylated mutant 439C/CT-M without a His-tag and 150 ng of streptavidin was added to the cis compartment. The record shows two channels that opened and stayed in the higher-conductance state (37–38 pS) at +50 mV. The solution on both sides of the membrane and filtering were the same as in Fig. 5 B.
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Figure 8: C domain with its amino terminus anchored on the cis side by streptavidin made normal channels. A preincubated mixture containing 4 ng of biotinylated mutant 439C/CT-M without a His-tag and 150 ng of streptavidin was added to the cis compartment. The record shows two channels that opened and stayed in the higher-conductance state (37–38 pS) at +50 mV. The solution on both sides of the membrane and filtering were the same as in Fig. 5 B.

Mentions: If the T and R domains of whole colicin Ia normally serve to anchor the amino-terminal end of the C domain on the cis side, then it should be possible to reconstitute this function in the isolated C domain by using streptavidin as a substitute anchor. Thus, a biotinylated carboxy-terminal fragment of colicin Ia (either 439C/CT-M or 381C/CT-L) was preincubated with streptavidin before the mixture was added to the cis compartment. The resulting single channels showed conductance and gating properties characteristic of whole colicin Ia channels (Fig. 8). (With biotinylated 439C/CT-M, channels that dropped to a low-conductance state were still sometimes observed; probably these arose from a small amount of unbiotinylated C domain remaining after purification on the monomeric avidin column, or else from biotinylated C domain that for some reason was not bound by streptavidin. Low-conductance channels appeared more rarely with biotinylated 381C/CT-L, which was first incubated with streptavidin before purification on a Superdex G-75 sizing column.) As expected, trans trypsin made the streptavidin-preincubated, biotinylated 439C/CT-M channels drop to a low-conductance state (data not shown).


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)

C domain with its amino terminus anchored on the cis side by streptavidin made normal channels. A preincubated mixture containing 4 ng of biotinylated mutant 439C/CT-M without a His-tag and 150 ng of streptavidin was added to the cis compartment. The record shows two channels that opened and stayed in the higher-conductance state (37–38 pS) at +50 mV. The solution on both sides of the membrane and filtering were the same as in Fig. 5 B.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230624&req=5

Figure 8: C domain with its amino terminus anchored on the cis side by streptavidin made normal channels. A preincubated mixture containing 4 ng of biotinylated mutant 439C/CT-M without a His-tag and 150 ng of streptavidin was added to the cis compartment. The record shows two channels that opened and stayed in the higher-conductance state (37–38 pS) at +50 mV. The solution on both sides of the membrane and filtering were the same as in Fig. 5 B.
Mentions: If the T and R domains of whole colicin Ia normally serve to anchor the amino-terminal end of the C domain on the cis side, then it should be possible to reconstitute this function in the isolated C domain by using streptavidin as a substitute anchor. Thus, a biotinylated carboxy-terminal fragment of colicin Ia (either 439C/CT-M or 381C/CT-L) was preincubated with streptavidin before the mixture was added to the cis compartment. The resulting single channels showed conductance and gating properties characteristic of whole colicin Ia channels (Fig. 8). (With biotinylated 439C/CT-M, channels that dropped to a low-conductance state were still sometimes observed; probably these arose from a small amount of unbiotinylated C domain remaining after purification on the monomeric avidin column, or else from biotinylated C domain that for some reason was not bound by streptavidin. Low-conductance channels appeared more rarely with biotinylated 381C/CT-L, which was first incubated with streptavidin before purification on a Superdex G-75 sizing column.) As expected, trans trypsin made the streptavidin-preincubated, biotinylated 439C/CT-M channels drop to a low-conductance state (data not shown).

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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