Use of synthetic gene technology resulted in the ability to produce MSPs in E. Modification of the MSP sequence subsequently allowed incorporation of affinity tags (Histidine, FLAG, etc.), which could be removed by protease treatment (Factor X, TEV) as well as provisions for site-specific labeling of engineered unique amino acid types. Realizing homogeneous and monodisperse particles as an endpoint required extensive protein engineering of the encircling Apo-AI protein, resulting in a class of amphipathic peptides termed membrane scaffold proteins (MSPs) ( Bayburt et al., 2002) and the corresponding discoidal bilayer particles with 10–12 nm diameter and 4.5–5.5 nm thickness, termed Nanodiscs ( Bayburt et al., 2002 Denisov et al., 2004). This process results in the membrane protein target being in its native-like lipid environment with ‘naturalistic presentation’, yet soluble in aqueous solution ( Sligar, 2003 Nath et al., 2007). While important to human diseases such as atherosclerosis, it was noted nearly a decade ago that the discoidal form of HDL particles could be utilized to provide a soluble supported phospholipid bilayer for the incorporation of membrane proteins ( Bayburt et al., 1998 2000 Bayburt and Sligar, 2002 2003 Civjan et al., 2003).
Consisting of primarily a single protein, Apo-AI, and lipid, these nanoscale particles transform from a lipid optimized discoidal form to a spherical ball as cholesterol ester is loaded ( Koppaka, 2001 Marcel and Kiss, 2003 Zannis et al., 2006). Important players in this process are the high-density lipoproteins. One example, which plays a critical role in human health, is the collection of lipid and protein particles which transport cholesterol and cholesterol esters between the vasculature and organs such as liver and steroidogenic tissues. The resulting cellular machines provide for the synthesis, transport and processing of information and energy. Self-assembly is the cornerstone of functioning biological systems, resulting in the synthesis of complex macromolecular assemblies containing protein, nucleic acid, lipid and carbohydrate components. We extensively characterize these structures by size exclusion chromatography and solution X-ray scattering. 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.
SCAFFOLD PROTEIN ASSEMBLY SERIES
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. The discoidal form of HDL is stabilized in solution by two encircling belts of Apo-AI. 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. 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.
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