Conformational distributions of melittin in water/methanol mixtures from frequency-domain measurements of nonradiative energy transfer.
We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal alpha-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 A. Increasing concentrations of methanol, which were shown to induce and alpha-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 A at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 A, as assessed from the center of the distance distribution. A distance of 24-25 A was recovered from the X-ray crystal structure of the tetramer, which is largely alpha-helical. At low ionic strength (less than 0.01) the CD spectra revealed a small fraction or amount of alpha-helix for melittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.
Lakowicz
J R
JR
University of Maryland School of Medicine, Department of Biological Chemistry, Baltimore 21201.
Gryczynski
I
I
Wiczk
W
W
Laczko
G
G
Prendergast
F C
FC
Johnson
M L
ML
D008555:Melitten / Q000737:chemistry*; D000432:Methanol; D011487:Protein Conformation; D012997:Solvents; D013050:Spectrometry, Fluorescence
D012997:Solvents; D008555:Melitten; D000432:Methanol