TY - JOUR
T1 - Conformational distributions of melittin in water / methanol mixtures from frequency-domain measurements of nonradiative energy transfer
AU - Lakowicz, Joseph R.
AU - Gryczynski, Ignacy
AU - Wiczk, Wieslaw
AU - Laczko, Gabor
AU - Prendergast, Franklyn C.
AU - Johnson, Michael L.
N1 - Funding Information:
This work was supportedb y Grant GM 35154 from the National Instituteso f Health,w ith support for instrumentationfr om the Natianal Sci-enceF oundation( DMB 8502835a nd 8511065t)o J.R.L., by Grant GM 34847f rom the National Instituteso f Healtht o F.G.P., and from theC enter for FluorescencSep ectroscopayt theU niversityo f Maryland.T he authorst hank Mr. Paul Yadlow-sky, University of Virginia, for assistancew ith measuringd istancesf rom the crystal structure. J.R.L. andG .L. wisht o acknowledgseu pportf rom the Medical BiotechnologyC enter,U niversityo f Maryland.
PY - 1990/7
Y1 - 1990/7
N2 - 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 α-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 Å. Increasing concentrations of methanol, which were shown to induce an α-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 Å 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 Å, as assessed from the center of the distance distribution. A distance of 24-25 Å was recovered from the X-ray crystal structure of the tetramer, which is largely α-helical. At low ionic strength ( < 0.01) the CD spectra revealed a small fraction or amount of α-helix for molittin 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.
AB - 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 α-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 Å. Increasing concentrations of methanol, which were shown to induce an α-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 Å 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 Å, as assessed from the center of the distance distribution. A distance of 24-25 Å was recovered from the X-ray crystal structure of the tetramer, which is largely α-helical. At low ionic strength ( < 0.01) the CD spectra revealed a small fraction or amount of α-helix for molittin 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.
KW - Distance distribution
KW - Energy transfer
KW - Fluorescence spectroscopy
KW - Frequency-domain fluorescence
KW - Melittin
KW - Time-resolved fluorescence
UR - http://www.scopus.com/inward/record.url?scp=0025369229&partnerID=8YFLogxK
U2 - 10.1016/0301-4622(90)85014-W
DO - 10.1016/0301-4622(90)85014-W
M3 - Article
C2 - 2207280
AN - SCOPUS:0025369229
SN - 0301-4622
VL - 36
SP - 99
EP - 115
JO - Biophysical Chemistry
JF - Biophysical Chemistry
IS - 2
ER -