The uncoupling protein from brown adipose tissue mitochondria. The environment of the tryptophan residues as revealed by quenching of the intrinsic fluorescence.
The uncoupling protein from brown adipose tissue is a member of the family of metabolite carriers of the mitochondrial inner membrane. It contains two tryptophan residues which have been characterized by fluorescence spectroscopy. Application of fluorescence-quenching-resolved spectroscopy (FQRS) allowed the determination of the emission maximum for each residue, both of which occur at 332 nm, thus suggesting that they are both located in a non-polar environment. Fluorescence quenching has demonstrated that both residues are accessible to acrylamide and inaccessible to Cs+, while only one of them is accessible to I-. When FQRS is combined with guanidinium hydrochloride denaturation, the unfolding of the regions containing each tryptophan can be monitored separately as they are transferred to the polar medium where the emission maximum appears at 359 nm, revealing also that the iodide-accessible residue is more sensitive to the denaturant. Secondary structure predictions, together with the data presented here, suggest that the iodide-accessible residue could correspond to Trp173 and the denaturant-resistant iodide-inaccessible one to Trp280, located in the center of the sixth transmembrane alpha-helix. Interaction of the protein with GDP (a transport inhibitor) has been studied and has revealed that it partially shields Trp173 from the interaction with I-, as well as reducing the static component of the acrylamide quenching.
Viguera
A R
AR
Department of Biochemistry, University of the Basque Country, Bilbao, Spain.
Goñi
F M
FM
Rial
E
E
D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D006224:Cricetinae; D019791:Guanidine; D006146:Guanidines; D006851:Hydrochloric Acid; D007473:Ion Channels; D008433:Mathematics; D008565:Membrane Proteins / Q000737:chemistry*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan; D000071256:Uncoupling Protein 1; D014508:Urea
D002352:Carrier Proteins; D006146:Guanidines; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D000071256:Uncoupling Protein 1; D014364:Tryptophan; D014508:Urea; D019791:Guanidine; D006851:Hydrochloric Acid