Characterization of apolipoprotein A-I structure using a cysteine-specific fluorescence probe

Area of Science:

• Biochemistry
• Structural Biology
• Lipid Metabolism

Background:

• Human apolipoprotein A-I (apoA-I) is crucial for high-density lipoprotein (HDL) structure and function.
• Understanding apoA-I's structural dynamics in lipid-free and lipid-bound states is key to its biological roles.
• Previous studies suggest complex structural organization, but detailed dynamics of its terminal regions require further investigation.

Purpose of the Study:

• To investigate the structural organization and dynamic properties of the N- and C-terminal regions of human apoA-I.
• To examine the effects of mutations and fluorescent labeling on apoA-I structure in lipid-free and lipid-bound states.
• To determine the spatial proximity of apoA-I's N- and C-termini in solution and in reconstituted HDL (rHDL).

Main Methods:

• Creation and expression of two Cys mutants of proapolipoprotein A-I (D9C and A232C) in E. coli.
• Site-specific fluorescent labeling using 6-acryloyl-2-dimethylaminonaphthalene (acrylodan).
• Spectroscopic analyses including circular dichroism, fluorescence spectroscopy, and Förster Resonance Energy Transfer (FRET).

Main Results:

• Mutations D9C and A232C did not affect apoA-I structure or stability in lipid-free or rHDL states.
• Acrylodan labeling did not alter apoA-I properties in lipid-bound states or the A232C mutant, but affected the D9C mutant's local stability.
• Fluorescence data confirmed a structured N-terminus and suggested a 3D C-terminus stabilized by protein contacts; FRET indicated N- and C-termini are near each other in rHDL.

Conclusions:

• The N-terminal region of apoA-I is well-organized, and the C-terminal region adopts a 3D structure.
• Protein folding in solution brings the C-terminus close to N-terminal Trp residues.
• In rHDL particles, the N- and C-terminal domains of apoA-I are in close proximity.