Spectral heterogeneity in protein fluorescence of bacteriorhodopsin: evidence for intraprotein aqueous regions

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Abstract
By the use of derivative spectral analysis, the broad tryptophan (Trp) fluorescence emission from aqueous suspensions of [Halobacterium halobium] bacteriorhodopsin in its native purple membrane may be resolved into contributions from buried, surface and exposed residues. Addition of glycerol produces a progressive enhancement of the fluorescence yield to a limiting value at about 70% vol/vol glycerol. Glycerol enhancement of fluorescence is also observed for monomeric Trp, and a good correlation exists between this effect and literature estimates of the fractional degree of Trp exposure in 9 globular proteins. The estimate of fractional Trp exposure in bacteriorhodopsin from this correlation (50 .+-. 15%) is in agreement with the value obtained by spectral differentiation and also by modified Stern-Volmer curves for quenching by water-soluble acrylamide. The absence of significant quenching by Tb(III) or Eu(III) ions, which may be expected to bind to the purple membrane surface, shows that the exposed Trp residues are in contact with water in intraprotein regions of the membrane and may be the 1st direct evidence for a transmembrane aqueous channel by which protons are actively transported during the bacteriorhodopsin photochemical cycle.