Biology subjects

Ludlam, W. G.

Publications and source records attributed to Ludlam, W. G..

3 recordsLinked to original sources

bioRxiv2024

DeFrND: detergent-free reconstitution into native nanodiscs with designer membrane scaffold peptides

Membrane scaffold proteins-based nanodiscs (NDs) have facilitated unprecedented structural and biophysical analysis of membrane proteins in a near-native lipid environment. However, successful reconstitution of membrane proteins in NDs requires prior solubilization and purification in detergents, which may impact their physiological structure and function. Furthermore, the detergent-mediated reconstitution of NDs is unlikely to recapitulate the precise composition or asymmetry of native membranes. To circumvent this fundamental limitation of traditional ND technology, we herein describe the development of membrane-solubilizing peptides to directly extract membrane proteins from native cell membranes into nanoscale discoids. By systematically protein engineering and screening, we created a new class of chemically modified Apolipoprotein-A1 mimetic peptides to enable the formation of detergent-free NDs (DeFrNDs) with high efficiency. NDs generated with these engineered membrane scaffold peptides are suitable for obtaining high-resolution structures using single-particle cryo-EM with native lipids. To further highlight the versatility of DeFrNDs, we directly extract a sampling of membrane signaling proteins with their surrounding native membranes for biochemical and biophysical interrogations.

biophysics↗

bioRxiv2023

Visualizing the chaperone-mediated folding trajectory of the G protein β5 β-propeller

The cytosolic Chaperonin Containing Tailless polypeptide 1 (CCT) complex is an essential protein folding machine with a diverse clientele of substrates, including many proteins with {beta}- propeller domains. Here, we determined structures of CCT in complex with its accessory co chaperone, phosducin-like protein 1 (PhLP1), in the process of folding G{beta}5, a component of Regulator of G protein Signaling (RGS) complexes. Cryo-EM and image processing revealed an ensemble of distinct snapshots that represent the folding trajectory of G{beta}5 from an unfolded molten globule to a fully folded {beta}-propeller. These structures reveal the mechanism by which CCT directs G{beta}5 folding through initiating specific intermolecular contacts that facilitate the sequential folding of individual {beta}-sheets until the propeller closes into its native structure. This work directly visualizes chaperone-mediated protein folding and establishes that CCT directs folding by stabilizing intermediates through interactions with surface residues that permit the hydrophobic core to coalesce into its folded state.

biochemistry↗

bioRxiv2019

Molecular Architecture of the Bardet-Biedl Syndrome 2-7-9 Subcomplex

Bardet-Biedl syndrome (BBS) is a genetic disease caused by mutations that disrupt the function of the BBSome, an eight-subunit complex that plays an important role in transport of proteins in primary cilia. To better understand the molecular basis of the disease, we analyzed the structure of a BBSome subcomplex consisting of three homologous BBS proteins (BBS2, BBS7, and BBS9) by an integrative structural modeling approach using electron microscopy and chemical crosslinking coupled with mass spectrometry. The resulting molecular model revealed an overall structure that resembles a flattened triangle. Within the structure, BBS2 and BBS7 form a tight dimer based on a coiled-coil interaction, and BBS9 associates with the dimer via an interaction with the -helical domain of BBS2. Interestingly, a BBS-linked mutation of BBS2 (R632P) is located in the -helical domain at the interface between BBS2 and BBS9, and binding experiments showed that this mutation disrupted the interaction of BBS2 with BBS9. This finding suggests that BBSome assembly is disrupted by the R632P substitution, providing a molecular explanation for BBS in patients harboring this mutation.

biochemistry↗