Abstract
High-density lipoproteins (HDLs) play an important role in human health through the metabolism and trafficking of cholesterol as well as providing the feedstocks for steroid hormone biosynthesis. These particles contain proteins, primarily Apo-AI and phospholipid and progress through various structural forms including 'lipid-poor', 'discoidal' and 'spherical' entities as cholesterol esters and lipid are incorporated. The discoidal form of HDL is stabilized in solution by two encircling belts of Apo-AI. Previous protein engineering of the Apo-AI sequence has led to a series of amphipathic helical proteins, termed membrane scaffold proteins (MSPs), which have shown great value in assembling nanoscale soluble membrane bilayers, termed Nanodiscs, of homogeneous size and composition and in the assembly of numerous integral membrane proteins for biophysical and biochemical investigations. In this communication we document a protein engineering approach to generate and optimize an extended polypeptide MSP, which will self-assemble phospholipids into larger Nanodiscs with diameters of 16-17 nm. We extensively characterize these structures by size exclusion chromatography and solution X-ray scattering.
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