Tryptophan fluorescence of mitochondrial uncoupling protein.
Mitochondrial uncoupling protein (UcP) contains two tryptophans buried in transmembrane alpha-helices: Trp-173 at the matrix end of fourth alpha-helix and Trp-280 on the sixth alpha-helix. However, the steady-state emission of isolated UcP exhibited properties unusual for alpha-helices: maximum close to that of free tryptophan emission and low quantum yield of 0.04. The former suggests prevailing tryptophan contacts with hydrophilic residues and confirms Trp-173 proximity to the water/membrane interface and Trp-280 location near a water-filled nucleotide-binding-site cavity. The latter might indicate that transmembrane segments are not true alpha-helices. Measured depolarization factor of 0.6 suggests also their "breathing". Analysis of UcP emission decays, measured by time-correlated-single-photon-counting, yielded components 0.4-0.6 ns, 2.2-3 ns and 9-10 ns (or alternatively 0.1, 1.5, 4.3 and 12.2 ns; or 0.1-0.3, 1.2, 3.7 and 10.5 ns), very similar to those of free tryptophan in water, where the longest component belongs to anionic form. Hence, such an "anionic" conformation must exist in UcP, perhaps as a consequence of charge-transfer complexes between Trp-173 & Lys-174 and Trp-280 & Arg-276. Moreover, N-ethylmaleimide modification, known to induce conformational changes, prolonged the "10 ns" component, decreased quantum yield to 0.03 without changes in emission spectra, while slightly shifting absorption to red and increasing tyrosine exposure to water.
Jezek
P
P
Department of Membrane Transport Biophysics, Academy of Sciences of the Czech Republic, Prague, Czech Republic. jezek@sun1.biomed.cas.cz
Lillo
P
P
Polecha
J
J
D002001:Adipose Tissue, Brown / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002352:Carrier Proteins / Q000737:chemistry*; D006224:Cricetinae; D005033:Ethylmaleimide / Q000494:pharmacology; D005454:Fluorescence Polarization; D066298:In Vitro Techniques; D007473:Ion Channels; D008565:Membrane Proteins / Q000737:chemistry*; D008647:Mesocricetus; D008928:Mitochondria / Q000737:chemistry; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014475:Uncoupling Agents / Q000737:chemistry; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; D014364:Tryptophan; D005033:Ethylmaleimide