Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV.
We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.
Weinberg
R B
RB
Department of Medicine, University of Texas Health Science Center, Houston 77225.
D001054:Apolipoproteins A / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013774:Tetranitromethane / Q000494:pharmacology; D014364:Tryptophan; D014443:Tyrosine
D001054:Apolipoproteins A; C022205:apolipoprotein A-IV; D014443:Tyrosine; D014364:Tryptophan; D013774:Tetranitromethane