Characterization of apolipoprotein A-I structure using a cysteine-specificfluorescence probe

Two new Cys mutants of proapolipoprotein A-I, D9C and A232C, were created a nd expressed in Escherichia coli systems. Specific labeling with the thiol- reactive fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (acrylod an), was used to study the structural organization and dynamic properties o f the extreme regions of human apolipoprotein A-I (apoA-I) in lipid-free an d lipid-bound states. Spectroscopic approaches, including circular dichrois m and various fluorescence methods, were used to examine the properties of the mutant proteins and of their covalent adducts with the fluorescence pro be. The mutations themselves had no effect on the structure and stability o f apoA-I in the lipid-free state and in reconstituted HDL (rHDL) complexes. Furthermore, covalent modification with acrylodan did not alter the proper ties of the apoA-I variants in the lipid-bound state nor in the lipid-free A232C mutant, but it affected the structure and local stability of the lipi d-free protein in the D9C mutant. Fluorescence results using the acrylodan probe confirmed a well-organized structure in the N-terminal region of apoA -I, Also, they suggested a three-dimensional structure in the C-terminal re gion, stabilized by protein-protein contacts. When Trp residues and acrylod an were used as donor-acceptor pairs for fluorescence resonance energy tran sfer (FRET), average distances could be measured. Both intensity and lifeti me changes of the Trp emission indicated a protein folding in solution that brings the C-terminus of the protein near the Trp residues in the N-termin al half of the sequence. Also, the N- and C-terminal domains of apoA-I appe ared to be near each other in rHDL having two apoA-I per particle.