CONFORMATIONAL-ANALYSIS OF GALANIN USING END-TO-END DISTANCE DISTRIBUTION OBSERVED BY FORSTER RESONANCE ENERGY-TRANSFER

The structural dynamics of the flexible neuropeptide galanin in soluti on were studied by Forster resonance energy transfer measurements at d ifferent temperatures by time-resolved fluorescence spectroscopy to de termine its conformational heterogeneity. Endogenous tryptophan at pos ition 2 acted as the fluorescent donor and the non fluorescent accepto r dinitrophenyl or the fluorescent acceptor dansyl were selectively at tached to lysine 25 in porcine galanin. The coexistence of different s tructures of the neuropeptide galanin in trifluoroethanol solution was revealed by the model independent analysis of the distribution of rel axation times from the time-resolved resonance energy transfer data. M ultiple conformational states are reflected by distinct end-to-end dis tance populations. The conformations differ in mean donor-acceptor dis tance by about 15 Angstrom, and are consistent with the extended and f olded backbone conformations of two alpha-helical regions separated by a flexible hinge. The effect that the labelling of galanin has on bin ding to the receptor was also evaluated. DNP-galanin showed the same h igh affinity to galanin receptors as unlabelled galanin, whereas DNS-g alanin had significantly reduced affinity.