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.

The Structure of Apolipoprotein A-I in High Density Lipoproteins

“…Additionally, the crystal structure shows that the four tryptophan residues present in the N terminus of apoA-I are surprisingly exposed to solvent, particularly Trp-8 and Trp-50. This is in contrast to fluorescence data obtained with monomeric apoA-I under physiological buffer conditions (30) showing a hydrophobic environment for these residues. Finally, our analysis of the crystal structure shows that the C-terminal portion of the helical bundle exhibits significant hydrophobic contacts, consistent with a stable bundle.…”

Impact of Self-association on Function of Apolipoprotein A-I

“…2A). The estimated ␣-helical content of WT[High] (ϳ60%) was similar to previously reported values under the same conditions (15,61), whereas the ϳ82% ␣-helical content of ⌬W[High] was significantly higher and comparable with the ␣-helicity of apoA-I on reconstituted discoidal HDL (15,61,62).…”

“…S4) (67,74). Consistent with this observation, solvent accessibility and Trp fluorescence measurements of monomeric lipid-free apoA-I demonstrate that the tertiary structure folding provides a hydrophobic environment for all four tryptophans (62,74,75). This suggests that tryptophans are directly involved in interhelical interactions within the fourhelix bundle.…”

The Roles of Tryptophans in Primer Synthesis by the DNA Primase of Bacteriophage T7

“…Different from hydrogen bond interactions, these hydrophobic interactions can be effective in a longer range, up to 10 nm (33). Tryptophan has shown to play a major role in the folding of a protein (20) and its stabilization (17,(21)(22)(23).…”

“…In another case, replacement of Trp-112 with phenylalanine in diacylglycerol kinase of E. coli reduces its expression level and its stability (17). Other studies also suggest that tryptophan plays a major role in the stabilization of proteins (21)(22)(23).…”