Major transmembrane movement associated with colicin Ia channel gating.

Abstract

Colicin Ia, a bacterial protein toxin of 626 amino acid residues, forms voltagedependent 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 resid:~es 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 disulfide 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.

Effect of Sequence Hydrophobicity and Bilayer Width upon the Minimum Length Required for the Formation of Transmembrane Helices in Membranes

“…The λmax values for peptides with 13 hydrophobic residues (pL 12 , p(LA) 6 and pL 6 A 6 ) also follow patterns somewhat analogous to that observed for sequences with 15 or 17 hydrophobic residues. However, the peptides with 13 hydrophobic residues exhibited much more red-shifted Trp fluorescence ( Figure 2C), than observed for peptides with 15 or 17 hydrophobic residues, and this, plus λmax vs. bilayer width profiles, were indicative of even less stable TM insertion and shorter TM segments than for the peptides with 15 or 17 hydrophobic residues.…”

“…In DOPC vesicles, pL 12 , p(LA) 6 , and pL 6 A 6 show high Q-ratios and large quencher induced λmax shifts, indicating a large amount of the non-TM state is present, ( Figure 2C). The lesser stability of the TM state for pL 12 relative to pL 14 and pL 16 , demonstrated by its exhibiting a high Q-ratio ( Figure 3A) and large quencher-induced λmax shifts in DOPC vesicles (Table 2), is particularly striking.…”

Effects of Membrane Potential and Sphingolipid Structures on Fusion of Semliki Forest Virus

“…The fusion protein of vesicular stomatitis virus also has an internal fusion peptide (63), and ion substitution experiments, similar to those performed for SFV (20), indicate that a trans-negative potential promotes fusion (1). For some bacterial toxins, notably colicins, hydrophobic internal segments spontaneously insert as a hairpin into a target membrane at low pH and then a trans-negative potential drives other segments of the protein into the membrane (23,41,48). Thus, it is possible that this common process used by bacteria to insert toxins into membranes also occurs in a viral system, that of SFV E1 fusion.…”

Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: Relevance to trypanosome lysis

“…Bilayers were formed at room temperature from soybean asolectin [lecithin type IIS (Sigma Chemical) from which nonpolar lipids had been removed (46)] across an 80-to 120-μm hole in a Teflon partition separating two aqueous solutions of 1 mL vol, as described previously (47). One percent asolectin in pentane (20 μL) was layered on top of both cis and trans solutions; the hole was pretreated with 3% (vol/vol) squalene (3 μL, Sigma) in petroleum ether, and the solvent was allowed to evaporate.…”