Apolipoprotein (apo)A-I is an organizing scaffold protein that is critical to high density lipoprotein (HDL) structure and metabolism, likely mediating many of its cardioprotective properties. However, HDL biogenesis is poorly understood as lipid-free apoA-I has been notoriously resistant to high resolution structural study. Published models from low resolution techniques share certain features but vary considerably in shape and secondary structure. To tackle this central issue in lipoprotein biology, we assembled an unprecedented team of lipoprotein structural biologists and set out to build a consensus model of monomeric lipid-free human apoA-I. Combining novel and published cross-link constraints, small angle X-ray scattering (SAXS), hydrogen-deuterium exchange (H-DX) and crystallography data, we propose a time averaged model consistent with much of the experimental data published over the last 40 years. The model provides a long sought platform for understanding and testing details of HDL biogenesis, structure and function.
APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: i) engineering a fusion protein construct in a codon optimized expression vector, ii) optimizing an efficient refolding protocol, and iii) screening buffer systems at physiological pH. The result was a high-yield (25 mg/l) bacterial expression system that produces lipid-free APOA5 soluble at concentrations of up to 10 mg/ml at a pH of 7.8 in bicarbonate buffers. Physical characterization of lipid-free APOA5 indicated that it exists as an array of multimers in solution, and far UV circular dichroism analyses show differences in total α-helicity between acidic and neutral pH buffering conditions. The protein was functional in that it bound and emulsified multilamellar dimyristoyl-phosphatidylcholine vesicles and could inhibit postprandial plasma TG accumulation when injected into C57BL/6J mice orally gavaged with Intralipid.
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