Production and Structural Analysis of Membrane‐Anchored Proteins in Phospholipid Nanodiscs

Abstract

Structural studies on membrane-anchored proteins containing a transmembrane (TM) helix have been hampered by difficulties in producing these proteins in a natively folded form. Detergents that are required to solubilize the hydrophobic TM helix usually destabilize the soluble domain. Thus, TM helices are removed for structural studies, which neglects the pivotal role of a membrane on protein function. This work presents a versatile strategy for the production of this protein class attached to phospholipid nanodiscs. By inserting the TM-helix into nanodiscs and a subsequent SortaseA-mediated ligation of the soluble domain, membrane-anchored BclxL could be obtained in a folded conformation. This strategy is suitable for high-resolution structure determination as well as for probing membrane location by NMR. This method will be applicable to a wide range of membrane-anchored proteins and will be useful to decipher their functional role in a native membrane environment.

The Structural Biology of Bcl-xL

“…All structures of Bcl-x L described above lack the C-terminal transmembrane domain due to difficulties producing the full-length protein in a soluble form at concentrations required for structural studies. This technical issue has, therefore, precluded structural studies on the membrane-bound form of Bcl-x L. However, two recent structural studies have begun to address the important question of how membranes affect the structure and function of pro-survival proteins given this is their natural cellular milieu [51,87,88]. Both studies use NMR-based techniques and employ nanodiscs to serve as a membrane surrogate.…”

Beyond detergent micelles: The advantages and applications of non-micellar and lipid-based membrane mimetics for solution-state NMR

“…In this approach, the soluble domain is never in contact with detergents and thus adopts a well-folded state in the full-length context. This functional feature was demonstrated by binding assays with canonical peptide ligand, which found binding affinities very similar to those obtained for the soluble domain alone [121]. The advantage of this approach is the possibility of segmental isotope labeling, which can be beneficial for visualizing the TMH in the full-length protein, since its resonances usually appear in the random-coil region and overlap with other signals originating from the soluble domain whose lines are much sharper due to faster tumbling outside the membrane.…”

“…In addition to detergent-based systems, TMHs have been inserted and structurally characterized in MSP nanodiscs. In recent years, the structure of the TMH of BclxL was investigated in DPC micelles [79] and phospholipid nanodiscs assembled with MSP1D1DH5 [121]. These studies showed that the structure of BclxL-TMH is very similar in both membrane mimetics.…”

“…For other membrane attached proteins that show a high degree of detergent sensitivity, the approach to solubilize the protein in detergent prior to nanodisc incorporation might be more difficult, calling for a detergent-free workflow of nanodisc assembly. For this scenario, a SortaseA-based method has been introduced for the production of full-length membrane-anchored proteins [121]. In this strategy, BclxL-TMH is produced separately, purified in detergent micelles and subsequently inserted into nanodiscs.…”

“…Furthermore, lipids with modified headgroups that contain a cysteineselective maleimide moiety can be used to produce membrane-anchored proteins, as demonstrated for Ras proteins[130], and probe their membrane location. Incorporation of paramagnetic Gd 3+ -labeled lipids is an excellent tool for deriving distance information between a membrane-attached protein, or selected parts of a MP, and the membrane surface[68,121,130]. MSP nanodiscs are the most suitable membrane mimetic for studying complexes of soluble proteins with an integral MP by NMR.…”