Scanning the Membrane-bound Conformation of Helix 1 in the Colicin E1 Channel Domain by Site-directed Fluorescence Labeling

Abdiwahab A Musse,Jie Wang,Gladys P Deleon,Gerry A Prentice,Erwin London,A Rod Merrill

doi:10.1074/jbc.m511140200

Abdiwahab A Musse, Jie Wang + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m511140200

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Abstract

Helix 1 of the membrane-associated closed state of the colicin E1 channel domain was studied by site-directed fluorescence labeling where bimane was covalently attached to a single cysteine residue in each mutant protein. A number of fluorescence properties of the tethered bimane fluorophore were measured in the membrane-bound state of the channel domain, including fluorescence emission maximum, fluorescence quantum yield, fluorescence anisotropy, membrane bilayer penetration depth, surface accessibility, and apparent polarity. The data show that helix 1 is an amphipathic alpha-helix that is situated parallel to the membrane surface. A least squares fit of the various data sets to a harmonic function indicated that the periodicity and angular frequency for helix 1 are typical for an amphipathic alpha-helix (3.7 +/- 0.1 residues per turn and 97 +/- 3.0 degrees, respectively) that is partially bathing into the membrane bilayer. Dual fluorescence quencher analysis also revealed that helix 1 is peripherally membrane-associated, with one face of the helix dipping into the lipid bilayer and the other face projecting toward the solvent. Finally, our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the membrane in support of a toroidal pore model for this colicin.

Highlights

Characterization of WT and Mutant p190H6—The N-terminal hexahistidine tag has been incorporated previously in the channel domain of colicin A and N, for ease of purification [30]
Structural and functional characterizations of His-tagged colicin A and N channel domains have shown that the incorporation of the hexahistidine tag did not significantly alter the channel properties of these two proteins [30]
Membrane binding analysis of the p190H6 revealed that the dissociation constant (Kd) for membrane association of the His-tagged channel peptide did not vary significantly from the untagged, thermolytic channel domain of WT colicin

Summary

Introduction

Characterization of WT and Mutant p190H6—The N-terminal hexahistidine tag has been incorporated previously in the channel domain of colicin A and N, for ease of purification [30]. Structural and functional characterizations of His-tagged colicin A and N channel domains have shown that the incorporation of the hexahistidine tag did not significantly alter the channel properties of these two proteins [30]. We used intrinsic Trp fluorescence as a probe of protein conformation as well as in vitro channel activity analysis to assess the impact of how the engineered hexahistidine tag, the introduction of cysteine residues, and the bimane labeling affected the structural and functional integrity of the protein. Membrane binding analysis of the p190H6 revealed that the dissociation constant (Kd) for membrane association of the His-tagged channel peptide did not vary significantly from the untagged, thermolytic channel domain of WT colici

Results

Discussion

Conclusion