Unraveling the mechanisms of tryptophan fluorescence quenching in the triosephosphate isomerase from Giardia lamblia.
In the native state several proteins exhibit a quenching of fluorescence of their tryptophans. We studied triosephosphate isomerase from Giardia lamblia (GlTIM) to dissect the mechanisms that account for the quenching of fluorescence of its Trp. GlTIM contains four Trp per monomer (Trp75, Trp162, Trp173, and Trp196) distributed throughout the 3D structure. The fluorescence of the denatured enzyme is 3-fold higher than that of native GlTIM. To ascertain the origin of this phenomenon, single and triple mutants of Trp per Phe were made. The intrinsic fluorescence was determined, and the data were interpreted on the basis of the crystal structure of the enzyme. Our data show that the fluorescence of all Trp residues is quenched through two different mechanisms. In one, fluorescence is quenched by aromatic-aromatic interactions due to the proximity and orientation of the indole groups of Trp196 and Trp162. The magnitude of the quenching of fluorescence in Trp162 is higher than in the other three Trp. Fluorescence quenching is also due to energy transfer to the charged residues that surround Trp 75, 173 and 196. Further analysis of the fluorescence of GlTIM showed that, among TIMs from other parasites, Trp at position 12 exhibits rather unique properties.
Hernández-Alcántara
Gloria
G
Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, 04530, México, D.F. gha@salud.gob.mx
Rodríguez-Romero
Adela
A
Reyes-Vivas
Horacio
H
Peon
Jorge
J
Cabrera
Nallely
N
Ortiz
Carmen
C
Enríquez-Flores
Sergio
S
De la Mora-De la Mora
Ignacio
I
López-Velázquez
Gabriel
G
D000818:Animals; D002942:Circular Dichroism; D004735:Energy Transfer / Q000502:physiology; D005453:Fluorescence; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
D050505:Mutant Proteins; D014364:Tryptophan; D014305:Triose-Phosphate Isomerase