Förster resonance energy transfer measurements are consistent with a helical bundle model for lipid-free apolipoprotein A-I.
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 Arg173 (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 approximately 22 A for W@50R83C and approximately 19 A 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 A, respectively, suggesting low conformational dynamics in both mutants. Differential scanning calorimetry (DSC) was performed on wt and a C-terminal deletion mutant, Delta(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.
Brouillette
Christie G
CG
Center for Biophysical Sciences and Engineering, Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. christie@uab.edu
Dong
Wen-Ji
WJ
Yang
Zhengrong W
ZW
Ray
Marjorie J
MJ
Protasevich
Irina I
II
Cheung
Herbert C
HC
Engler
Jeffrey A
JA
D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008958:Models, Molecular; D016296:Mutagenesis; D009282:Naphthalenesulfonates
D016632:Apolipoprotein A-I; D008055:Lipids; D009282:Naphthalenesulfonates; C006422:1,8-I-AEDANS