Apolipoprotein (apo) A-I mutants were constructed for FRET studies to distinguish between two possible lipid-free conformers, a globular helix bundle and an elongated helical hairpin. Mutants containing a single Trp at position 50 were prepared by replacing Trps at positions 8, 72, and 108 with Phe (W@50). Two mutants were constructed from W@50 by incorporating Cys at Arg83 (W@50R83C) or Arg 173 (W@50R173C) for attachment of the fluorescent probe AEDANS. Secondary structure of the mutants is very similar to wild type (wt) apo A-I, and fluorescence emission indicates that W50 is protected from solvent. Thermal stabilities of the AEDANS-labeled mutants are also similar to wt. These results indicate that no discernible changes occur in structure or stability as a result of mutations or labeling. The FRET data from W@50 to AEDANS are well-represented by a single distance distribution function with a distance of ∼22 Å for W@50R83C and ∼19 Å for W@50R173C. These distances are consistent with theoretical values calculated from a helical bundle model but not from a helical hairpin. A probability distance distribution function yields significantly small half-width values of 5.6 and 3.7 Å, respectively, suggesting low conformational dynamics in both mutants. Differential scanning calorimetry (DSC) was performed on wt and a C-terminal deletion mutant, Δ(187-243), to obtain information on domain architecture. Contrary to expectations, both proteins unfold cooperatively. The results are consistent with the presence of a single folded domain within residues 1-186. These results support the presence of a discrete globular bundle conformation for lipid-free apo A-I. © 2005 American Chemical Society.