The preference of tryptophan for membrane interfaces: insights from N-methylation of tryptophans in gramicidin channels.
To better understand the structural and functional roles of tryptophan at the membrane/water interface in membrane proteins, we examined the structural and functional consequences of Trp --> 1-methyl-tryptophan substitutions in membrane-spanning gramicidin A channels. Gramicidin A channels are miniproteins that are anchored to the interface by four Trps near the C terminus of each subunit in a membrane-spanning dimer. We masked the hydrogen bonding ability of individual or multiple Trps by 1-methylation of the indole ring and examined the structural and functional changes using circular dichroism spectroscopy, size exclusion chromatography, solid state (2)H NMR spectroscopy, and single channel analysis. N-Methylation causes distinct changes in the subunit conformational preference, channel-forming propensity, single channel conductance and lifetime, and average indole ring orientations within the membrane-spanning channels. The extent of the local ring dynamic wobble does not increase, and may decrease slightly, when the indole NH is replaced by the non-hydrogen-bonding and more bulky and hydrophobic N-CH(3) group. The changes in conformational preference, which are associated with a shift in the distribution of the aromatic residues across the bilayer, are similar to those observed previously with Trp --> Phe substitutions. We conclude that indole N-H hydrogen bonding is of major importance for the folding of gramicidin channels. The changes in ion permeability, however, are quite different for Trp --> Phe and Trp --> 1-methyl-tryptophan substitutions, indicating that the indole dipole moment and perhaps also ring size and are important for ion permeation through these channels.
Sun
Haiyan
H
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Greathouse
Denise V
DV
Andersen
Olaf S
OS
Koeppe
Roger E
RE
D002850:Chromatography, Gel; D002942:Circular Dichroism; D003903:Deuterium / Q000378:metabolism; D006096:Gramicidin / Q000737:chemistry / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008745:Methylation; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*
D007473:Ion Channels; D008565:Membrane Proteins; D006096:Gramicidin; C028129:tryptophan methyl ester; D014364:Tryptophan; D003903:Deuterium