title: Structural organization of the N-terminal domain of apolipoprotein A-I: studies of tryptophan mutants.
abstract: Site-directed mutagenesis and detailed fluorescence studies were used to study the structure and dynamics of recombinant human proapolipoprotein (proapo) A-I in the lipid free state and in reconstituted high-density lipoprotein (rHDL) particles. Five different mutants of proapoA-I, each containing a single tryptophan residue, were produced in bacteria corresponding to each of the naturally occurring Trp residues (position -3 in the pro-segment, 8, 50, 72, and 108) in the N-terminal half of the protein. Structural analyses indicated that the conservative Phe-Trp substitutions did not perturb the conformation of the mutants with respect to the wild-type protein. Steady-state fluorescence studies indicated that all of the Trp residues exist in nonpolar environments that are highly protected from solvent in both the lipid-free and lipid-bound forms. Time-resolved lifetime and anisotropy studies indicated that the shape of the monomeric form of proapoA-I is a prolate ellipsoid with an axial ratio of about 6:1. In addition, the region surrounding Trp 108 appears to be more mobile than the rest of the protein in the lipid-free state. However, in rHDL particles, no significant domain motion was detected for any of the Trp residues. The results presented in this work are consistent with a model for monomeric lipid-free proapoA-I in which the N-terminal half of the molecule is organized into a bundle of helices.
authors.affiliation: Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois 61801, USA.
authors.forename: W S
authors.initials: WS
authors.lastname: Davidson
authors.forename: K
authors.initials: K
authors.lastname: Arnvig-McGuire
authors.forename: A
authors.initials: A
authors.lastname: Kennedy
authors.forename: J
authors.initials: J
authors.lastname: Kosman
authors.forename: T L
authors.initials: TL
authors.lastname: Hazlett
authors.forename: A
authors.initials: A
authors.lastname: Jonas
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics*; D001054:Apolipoproteins A / Q000737:chemistry* / Q000235:genetics*; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics*
chemical_list: D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D018076:DNA, Complementary; D008075:Lipoproteins, HDL; D011498:Protein Precursors; D011994:Recombinant Proteins; D010649:Phenylalanine; D014364:Tryptophan