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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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Linear diagram showing the approximate lengths of whole colicin Ia and the four carboxy-terminal fragments, CT-S, CT-M, CT-L, and CT-XL. The boundaries of the T, R, and C domains are from Wiener et al. 1997. The inter-domain regions are shaded and the full length of helix 1 is indicated. Mutant fragments have a cysteine residue near the amino terminus (453C/CT-S, 439C/CT-M, 326C/CT-L, and “−3” C/CT-XL), or not so near it (381C/CT-L). The amino-terminal His-tags are not shown.
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Figure 2: Linear diagram showing the approximate lengths of whole colicin Ia and the four carboxy-terminal fragments, CT-S, CT-M, CT-L, and CT-XL. The boundaries of the T, R, and C domains are from Wiener et al. 1997. The inter-domain regions are shaded and the full length of helix 1 is indicated. Mutant fragments have a cysteine residue near the amino terminus (453C/CT-S, 439C/CT-M, 326C/CT-L, and “−3” C/CT-XL), or not so near it (381C/CT-L). The amino-terminal His-tags are not shown.

Mentions: Whole colicin Ia was prepared as previously described (Qiu et al. 1994). We prepared carboxy-terminal fragments of colicin Ia in four different lengths, designated CT short (CT-S; residues 453–626), CT medium (CT-M; 438–626), CT long (CT-L; 327–626), and CT extra long (CT-XL; 282–626) (Fig. 2), as follows. The shortest fragment, CT-S, was cloned by using site-directed mutagenesis to create a BamHI site overlapping the codons for amino acids 450–452 in the colicin Ia gene cloned in pUC19 (Jakes et al., 1998). The resulting plasmid was digested with BamHI and the fragment containing the 3′ end of the colicin Ia gene, beginning with the codon for Ile 453 and ending with residue Ile 626, as well as the Ia immunity gene, was ligated at the BamHI site of the expression vector pET-15b (Novagen, Inc.) to create pKSJ120. The sequence at the amino terminus of the resulting protein is thus: MGSSHHHHHHSSGLVPRGSHMLEDP I453N454… Residues shown in italics are from the pET vector His-tag and cloning sequences; the beginning of the colicin sequence is shown in bold.


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)

Linear diagram showing the approximate lengths of whole colicin Ia and the four carboxy-terminal fragments, CT-S, CT-M, CT-L, and CT-XL. The boundaries of the T, R, and C domains are from Wiener et al. 1997. The inter-domain regions are shaded and the full length of helix 1 is indicated. Mutant fragments have a cysteine residue near the amino terminus (453C/CT-S, 439C/CT-M, 326C/CT-L, and “−3” C/CT-XL), or not so near it (381C/CT-L). The amino-terminal His-tags are not shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Linear diagram showing the approximate lengths of whole colicin Ia and the four carboxy-terminal fragments, CT-S, CT-M, CT-L, and CT-XL. The boundaries of the T, R, and C domains are from Wiener et al. 1997. The inter-domain regions are shaded and the full length of helix 1 is indicated. Mutant fragments have a cysteine residue near the amino terminus (453C/CT-S, 439C/CT-M, 326C/CT-L, and “−3” C/CT-XL), or not so near it (381C/CT-L). The amino-terminal His-tags are not shown.
Mentions: Whole colicin Ia was prepared as previously described (Qiu et al. 1994). We prepared carboxy-terminal fragments of colicin Ia in four different lengths, designated CT short (CT-S; residues 453–626), CT medium (CT-M; 438–626), CT long (CT-L; 327–626), and CT extra long (CT-XL; 282–626) (Fig. 2), as follows. The shortest fragment, CT-S, was cloned by using site-directed mutagenesis to create a BamHI site overlapping the codons for amino acids 450–452 in the colicin Ia gene cloned in pUC19 (Jakes et al., 1998). The resulting plasmid was digested with BamHI and the fragment containing the 3′ end of the colicin Ia gene, beginning with the codon for Ile 453 and ending with residue Ile 626, as well as the Ia immunity gene, was ligated at the BamHI site of the expression vector pET-15b (Novagen, Inc.) to create pKSJ120. The sequence at the amino terminus of the resulting protein is thus: MGSSHHHHHHSSGLVPRGSHMLEDP I453N454… Residues shown in italics are from the pET vector His-tag and cloning sequences; the beginning of the colicin sequence is shown in bold.

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