Format

Send to

Choose Destination
Methods Mol Biol. 2019;1877:233-246. doi: 10.1007/978-1-4939-8861-7_16.

Reconstitution and Characterization of BCL-2 Family Proteins in Lipid Bilayer Nanodiscs.

Author information

1
Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
2
Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA. fmarassi@sbp.edu.

Abstract

The BCL-2 family proteins are key regulators of programmed cell death or apoptosis, and represent important targets for the development of anticancer drugs. Because their functions are intimately connected with intracellular membranes, it is important to perform structural and activity studies in precisely characterized samples that include phospholipids and capture the features of the native physiological environment as closely as possible. NMR studies and activity assays based on lipid bilayer nanodiscs are ideally suited for this purpose: they enable the conformations and interactions of these proteins to be probed at atomic resolution in their membrane-associated states. Here we describe detailed protocols for generating the protein components and the reconstituted nanodisc samples suitable for NMR studies and functional assays. The protocols focus on the BCL-2 family protein BCL-XL, a dominant inhibitor of programmed cell death and a major anticancer drug target. The protocols are relatively straightforward. Provided care is taken to ensure protein integrity and sample homogeneity, BCL-XL can be readily reconstituted in nanodiscs, with its hydrophobic C-terminal tail anchored through the nanodisc lipid bilayer, and its folded N-terminal head and ligand binding pocket exposed to the aqueous solution. We anticipate that BCL-2 samples prepared with these protocols will advance structural and mechanistic studies for this important protein family.

KEYWORDS:

Apoptosis; BCL-2; Membrane; NMR; Nanodisc; Structure

Supplemental Content

Full text links

Icon for Springer
Loading ...
Support Center