New insight into the self-association of human apolipoprotein A-I

Apolipoprotein A-I (apoA-I) is a critical plasma protein responsible for high-density lipoprotein formation, playing a vital role in reverse-cholesterol transport. Lipid-free apoA-I has two domains, an N-terminal helix bundle and a structurally less organized C-terminal (CT) region. In solution, apoA-I self-associates, which is mediated by the CT domain. To gain insight into the self-associated state, cysteine was introduced in each of the three putative α-helices of the CT domain: S201C for helix-8, Q216C for helix-9, and S231C for helix-10, and a S25C mutation served as a control. The single cysteine mutants were covalently labeled with pyrene, a spatially sensitive probe producing fluorescence excimers when in close proximity. At a protein concentration of 0.2 mg/mL, strong excimers were observed for S201C-pyrene-labeled apoA-I, while excimer intensity was weaker for Q216C- and S231C-pyrene-labeled apoA-I. When the protein was diluted 10-fold, pyrene excimer fluorescence was reduced, but excimer fluorescence remained strong for S201C-apoA-I, implying the protein remained in a self-associated state. Cysteine-specific crosslinking was more efficient for S201C compared to Q216C and S231C apoA-I mutants, in agreement with the pyrene excimer analysis. Size-exclusion chromatography demonstrated that at 0.02 mg/mL, apoA-I is present as a mixture of monomers and dimers, and therefore the observed pyrene excimers at 0.02 mg/mL are caused by dimerization of apoA-I. In the apoA-I dimer, helix-8 is more buried and positioned near a neighboring helix-8, while helices-9 and -10 are further apart and more exposed. This structural arrangement potentially results in an optimal position for helix-10 to engage in lipid binding.