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Structure in the channel forming domain of colicin E1 bound to membranes: The 402–424 sequence
Author(s) -
SalwiSki Ukasz,
Hubbell Wayne L.
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.3.562
Subject(s) - chemistry , helix (gastropod) , crystallography , colicin , lipid bilayer , transmembrane domain , membrane , sequence (biology) , side chain , protein secondary structure , bilayer , nitroxide mediated radical polymerization , electron paramagnetic resonance , biophysics , nuclear magnetic resonance , biochemistry , biology , dna , physics , ecology , radical polymerization , organic chemistry , snail , copolymer , plasmid , polymer
To explore the structure of the pore‐forming fragment of colicin E1 in membranes, a series of 23 consecutive single cysteine substitution mutants was prepared in the sequence 402–424. Each mutant was reacted with a sulfhydryl‐specific reagent to generate a nitroxide labeled side chain, and the mobility of the side chain and its accessibility to collision with paramagnetic reagents was determined from the electron paramagnetic resonance spectrum. Individual values of these quantities were used to identify tertiary contact sites and the nature of the surrounding solvent, while their periodic dependence on sequence position was used to identify secondary structure. In solution, the data revealed a regular helix of 11 residues in the region 406–416, consistent with helix IV of the crystal structure. Upon binding to negatively charged membranes at pH 4.0, helix IV apparently grows to a length of 19 residues, extending from 402–420. One face of the helix is solvated by the lipid bilayer, and the other by an environment of a polar nature. Surprisingly, a conserved charged pair, D408–R409, is located on the lipid‐exposed face. Evidence is presented to suggest a transmembrane orientation of this new helix, although other topographies may exist in equilibrium.