EFFECT OF GUANINE-NUCLEOTIDE-BINDING ON THE INTRINSIC TRYPTOPHAN FLUORESCENCE PROPERTIES OF RAB5

To gain further insight into structural elements involved in Rab5 func tion, differences in the intrinsic tryptophan fluorescence of the GDP- and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-bound forms of the protein were examined. When excited at 290 nm, Rab5 displays emiss ion maxima at 339.7 nm for the GDP-bound and 336.7 nm for the GTP gamm a S-bound forms. The tryptophan fluorescence intensity is quenched by similar to 25% in the GTP gamma S-bound form relative to the GDP-bound conformation. Variant Rab5 molecules were created by site-directed mu tagenesis to convert the protein's two tryptophans to phenylalanine re sidues. Fluorescence studies reveal that the observed changes upon GDP /GTP gamma S GTP gamma S exchange are due to a blue shift in the emiss ion spectra for both Trp(74) (342.0 to 339.5 nm) and Trp(114) (335.3 t o 333.7 nm) and fluorescence quenching of Trp(114). Consistent with th e blue shift in the emission spectra, both tryptophans are more resist ant to oxidation by N-bromosuccinimide in the GTP gamma S-bound state. These data indicate that both of Rab5's tryptophans are brought into a more sequestered, hydrophobic environment upon conformational change s promoted by guanine nucleotide exchange. Since Trp(74) lies adjacent to Rab5's cognate switch II domain, local conformational changes woul d be predicted based on the known structure of Ras. However, Trp(114) lies within a region of Rab5 potentially related to the switch III dom ain unique to heterotrimeric G(alpha t). Thus, changes in the fluoresc ence properties of Trp(114) upon guanine nucleotide exchange suggest t hat Rab proteins may have structure-function relationships similar to those described for heterotrimeric GTP-binding proteins.