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| Major transmembrane movement associated with colicin Ia channel gating |
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| Author(s): Qiu XQ, Jakes KS, Kienker PK, Finkelstein A, Slatin SL |
| Source: JOURNAL OF GENERAL PHYSIOLOGY Volume: 107 Issue: 3 Pages: 313-328 Published: MAR 1996 |
| Times Cited: 70 References: 45 |
| Abstract: Colicin Ia, a bacterial protein toxin of 626 amino acid residues, forms voltage-dependent channels in planar lipid bilayer membranes. We have exploited the high affinity binding of streptavidin to biotin to map the topology of the channel-forming domain (roughly 175 residues of the COOH-terminal end) with respect to the membrane. That is, we have determined, for the channel's open and closed states, which parts of this domain are exposed to the aqueous solutions on either side of the membrane and which are inserted into the bilayer. This was done by biotinylating cysteine residues introduced by site-directed mutagenesis, and monitoring by electrophysiological methods the effect of streptavidin addition on channel behavior. We have identified a region of at least 68 residues that flips back and forth across the membrane in association with channel opening and closing. This identification was based on our observations that for mutants biotinylated in this region, streptavidin added to the cis (colicin-containing) compartment interfered with channel opening, and trans streptavidin interfered with channel closing. (If biotin was linked to the colicin by a di-sulfide bond, the effects of streptavidin on channel closing could be reversed by detaching the streptavidin-biotin complex from the colicin, using a water-soluble reducing agent. This showed that the cysteine sulfur, not just the biotin, is exposed to the trans solution.) The upstream and downstream segments flanking the translocated region move into and out of the bilayer during channel opening and closing, forming two transmembrane segments. Surprisingly, if any of several residues near the upstream end of the translocated region is held on the cis side by streptavidin, the colicin still forms voltage-dependent channels, indicating that a part of the protein that normally is fully translocated across the membrane can become the upstream transmembrane segment. Evidently, the identity of the upstream transmembrane segment is not crucial to channel formation, and several open channel structures can exist. |
| Document Type: Article |
| Language: English |
Addresses:
1. ALBERT EINSTEIN COLL MED, DEPT PHYSIOL & BIOPHYS, BRONX, NY 10461 USA
2. ALBERT EINSTEIN COLL MED, DEPT NEUROSCI, BRONX, NY 10461 USA |
| Publisher: ROCKEFELLER UNIV PRESS, 222 E 70TH STREET, NEW YORK, NY 10021 |
| Subject Category: Physiology |
| IDS Number: UA394 |
| ISSN: 0022-1295 |
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