Apolipoprotein (apo) E serves as a ligand for the low-density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal (NT) domain of lipid-free apoE exists as globular four-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding induced conformational change. To investigate the structural basis for this phenomenon, site directed spin label electron paramagnetic resonance (EPR) spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala164). Using Cys112Ser apoE-NT as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176 and 181) were produced, isolated and labeled with the nitroxide probe, methane thiosulfonate. EPR analysis revealed that lipid association induces fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site directed fluorescence analysis using an environmentally sensitive probe indicated the lipid-induced transition of this region of the protein to alpha helix is accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induces an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE