In bacteria, certain shape-sensing proteins localize to differently curved membranes. During sporulation in Bacillus subtilis, the only convex (positively curved) surface in the cell is the forespore, an approximately spherical internal organelle. Previously, we demonstrated that SpoVM localizes to the forespore by preferentially adsorbing onto slightly convex membranes. Here, we used NMR and molecular dynamics simulations of SpoVM and a localization mutant (SpoVM P9A ) to reveal that SpoVM's atypical amphipathic α-helix inserts deeply into the membrane and interacts extensively with acyl chains to sense packing differences in differently curved membranes. Based on binding to spherical supported lipid bilayers and Monte Carlo simulations, we hypothesize that SpoVM's membrane insertion, along with potential cooperative interactions with other SpoVM molecules in the lipid bilayer, drives its preferential localization onto slightly convex membranes. Such a mechanism, which is distinct from that used by high curvature-sensing proteins, may be widely conserved for the localization of proteins onto the surface of cellular organelles.ArfGAP1 | SpoIVA | DivIVA | curvature sensing | lipid packing

Many essential cellular processes including endocytosis and vesicle trafficking require alteration of membrane geometry. These changes are usually mediated by proteins that can sense and/or induce membrane curvature. Using spherical nanoparticle supported lipid bilayers (SSLBs), we characterize how SpoVM, a bacterial development factor, interacts with differently curved membranes by magic angle spinning solid-state NMR. Our results demonstrate that SSLBs are an effective system for structural and topological studies of membrane geometry-sensitive molecules.

LR11 (SorLA) is a recently identified neuronal protein that interacts with amyloid precursor protein (APP), a central player in the pathology of the Alzheimer's disease (AD). AD is a neurodegenerative disease and the most common cause of dementia in the elderly. Current estimates suggest that as many as 5.3 million Americans are living with AD. Recent investigations have uncovered the pathophysiological relevance of APP intracellular trafficking in AD. LR11 is of particular importance due to its role in regulating APP transport and processing. LR11 is a type I transmembrane protein and belongs to a novel family of Vps10p receptors. Using a new expression vector, pMTTH, (MBP-MCS1(multiple cloning site)-Thrombin protease cleavage site-MCS2-TEV protease cleavage site-MCS3-His 6 ), we successfully expressed, purified and reconstituted the LR11 transmembrane (TM) and cytoplasmic (CT) domains into bicelles and detergent micelles for NMR structural studies. This new construct allowed us to overcome several obstacles during sample preparation. MBP fused LR11 TM and LR11TMCT proteins are preferably expressed at high levels in E. coli membrane, making a refolding of the protein unnecessary. The C-terminal His-tag allows for easy separation of the target protein from the truncated products from the C-terminus, and provides a convenient route for screening detergents to produce high quality 2D 1 H-15 N TROSY spectra. Thrombin protease cleavage is compatible with most of the commonly used detergents, including a direct cleavage at the E. coli membrane surface. This new MBP construct may provide an effective route for the preparation of small proteins with TM domains.