Conformational Transitions in the Membrane Scaffold Protein of Phospholipid Bilayer Nanodiscs

Abstract

Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipidfree and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest. Molecular & Cellular

Lipid-Mediated Regulation of Embedded Receptor Kinases via Parallel Allosteric Relays

“…The scaffold protein MSP1D1, which is essential for the integrity of nanodiscs, served as a suitable control to determine the effects of lipid composition on scaffold protein composition (34). We observed only minor effects of lipid composition on exchange across MSP1D1 peptides (Table S1).…”

Assessment of differences in the conformational flexibility of hepatitis B virus core‐antigen and e‐antigen by hydrogen deuterium exchange‐mass spectrometry

“…[16][17][18][19][20][21][22][23], in the a2b helix, and the junction with the a3 helix (peptide [38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55], and in the junction between a4b and a5 helices (peptide 104-116). Peptides were not identified for the central region of the a3 helix (residues [56][57][58][59][60][61][62][63][64][65][66][67][68] and the N-terminal half of the a5 helix (residues 121-125), hence the deuterium incorporation of these regions is unknown. Viewed in the context of the folded structure, the overall pattern of incorporation is clear [ Fig.…”

“…52,[58][59][60] Unfortunately, observation of EX1-type kinetics is often an artifact attributable either to chemical perturbation of the proteins, or to experimental error, such as carryover between injections. 61,62 Carryover was specifically avoided in our experiments by extensive washing of the pepsin and LC columns between sample injections (see Materials and Methods).…”

Replication in Bioanalytical Studies with Hdx MS: Aim as High as Possible

“…A second example of the value of biological replication comes from our HDX MS work with membrane proteins [7][8][9][10][11][12][13][14]. Depending on the batch of lipid mimetics (liposomes or nanodiscs), there can be variability in deuterium incorporation in membrane proteins.…”