A tryptophan rotamer located in a polar environment probes pH-dependent conformational changes in bovine beta-lactoglobulin A.
Bovine beta-lactoglobulin A (BLGA) is a well characterized globular protein whose tertiary structure has been investigated in detail. BLGA undergoes a pH-dependent conformational change which X-ray data described as involving mostly the loop connecting strands E and F and the deprotonation of a glutamic acid residue (Glu89). These structural changes have been investigated using, among other techniques, fluorescence spectroscopy. The intrinsic fluorescence of BLGA is dominated by two Trp residues. These residues are located far from the EF loop and would not be expected to probe the pH-induced conformational change of the protein. Trp19 is located at the bottom of the interior beta-barrel, whereas Trp61 is located at the aperture of the barrel near the CD loop and is "silent" in the emission of native BLGA because of the proximity of a disulfide moiety. Our study suggests that, surprisingly, the fluorescence of Trp19 has the characteristic of a more polar environment than structural models from X-ray data would suggest and that at least two distinct conformations (or rotamers) of Trp19 contribute to the fluorescence of the protein. The less populated rotamer (relative amplitude (alpha) approximately 20%, tau approximately 3 ns) probes a more polar environment and a pH-dependent conformational change of BLGA in the region of Trp19 which X-ray data do not detect. Finally, our study provides the estimate of the fluorescence lifetime of Trp61 in the "unquenched" form.
Harvey
Billie J
BJ
Department of Physics and Astronomy, University of Texas at San Antonio, San Antonio, Texas, USA.
Bell
Erin
E
Brancaleon
Lorenzo
L
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D000596:Amino Acids; D007782:Lactoglobulins; C016952:N-acetyltryptophanamide; D014364:Tryptophan; D014508:Urea