Aromatic interactions in homeodomains contribute to the low quantum yield of a conserved, buried tryptophan

Trp 48, a conserved, buried residue commonly found in the hydrophobic core of homeodomains, has an unusually low fluorescence quantum yield. Chemical denaturation of Drosophila homeodomains Engrailed and Antennapedia(C39S) re sult in a four-fold increase in quantum yield, while unfolding of Ultrabith orax causes a twenty-fold enhancement. Global analysis of time-resolved flu orescence decay monitored at multiple emission wavelengths reveals sub-nano second lifetime components which dominate the overall intensity, Based on s tructure and sequence analysis of several homeodomains, we deduce that quen ching is due to a transient, excited-state NH ... pi hydrogen bond involvin g Trp 48 and a conserved aromatic residue at position 8. Additionally, both time-resolved fluorescence of indole-benzene mixtures and an electrostatic model of the proposed tryptophan-aromatic interaction substantiate differe nt aspects of this mechanism. A survey of the Protein Data Bank reveals man y proteins with tryptophan-aromatic pairs where the indole nitrogen partici pates in a NH ... pi hydrogen bond with the ring of another aromatic residu e. Chemical denaturation of one protein found in this survey, human fibrone ctin type III module 10, causes an enhancement of the fluorescence quantum yield. This unique interaction has implications for many other systems and may be useful for studying larger, multi-tryptophan containing proteins. (C ) 2000 Wiley-Liss, Inc.