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

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