Effect of guanine nucleotide binding on the intrinsic tryptophan fluorescence properties of Rab5.
To gain further insight into structural elements involved in Rab5 function, 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 emission maxima at 339.7 nm for the GDP-bound and 336.7 nm for the GTP gamma S-bound forms. The tryptophan fluorescence intensity is quenched by approximately 25% in the GTP gamma S-bound form relative to the GDP-bound conformation. Variant Rab5 molecules were created by site-directed mutagenesis to convert the protein's two tryptophans to phenylalanine residues. Fluorescence studies reveal that the observed changes upon GDP/GTP gamma S exchange are due to a blue shift in the emission spectra for both Trp74 (342.0 to 339.5 nm) and Trp114 (335.3 to 333.7 nm) and fluorescence quenching of Trp114. Consistent with the blue shift in the emission spectra, both tryptophans are more resistant 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 changes promoted by guanine nucleotide exchange. Since Trp74 lies adjacent to Rab5's cognate switch II domain, local conformational changes would be predicted based on the known structure of Ras. However, Trp114 lies within a region of Rab5 potentially related to the switch III domain unique to heterotrimeric G alpha t. Thus, changes in the fluorescence properties of Trp114 upon guanine nucleotide exchange suggest that Rab proteins may have structure-function relationships similar to those described for heterotrimeric GTP-binding proteins.
Pan
J Y
JY
Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Sanford
J C
JC
Wessling-Resnick
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001974:Bromosuccinimide; D002793:Cholic Acids; D003902:Detergents; D020558:GTP Phosphohydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010084:Oxidation-Reduction; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan; D020696:rab5 GTP-Binding Proteins
D002793:Cholic Acids; D003902:Detergents; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014364:Tryptophan; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D001974:Bromosuccinimide; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate