Structural organization of the n-terminal domain of apolipoprotein A-I: Studies of tryptophan mutants

Site-directed mutagenesis and detailed fluorescence studies were used to st udy the structure and dynamics of recombinant human proapolipoprotein (proa po) A-I in the lipid free state and in reconstituted high-density lipoprote in (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 analys es indicated that the conservative Phe-Trp substitutions did not perturb th e 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 l ipid-free and lipid-bound forms, Time-resolved lifetime and anisotropy stud ies indicated that the shape of the monomeric form of proapoA-I is a prolat e ellipsoid with an axial ratio of about 6.1. In addition, the region surro unding Trp 108 appears to be more mobile than the rest of the protein in th e lipid-free state. However, in rHDL particles, no significant domain motio n was detected for any of the Trp residues. The results presented in this w ork are consistent with a model for monomeric lipid-free proapoA-I in which the N-terninal half of the molecule is organized into a bundle of helices.