Edited by Jan RydströmKeywords: ATP-binding cassette transporter Substrate-binding protein Ligand receptor Tripartite ATP-independent periplasmic transporter Structural classification a b s t r a c t Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth of structural and functional data is available on SBPs, with over 120 unique entries in the Protein Data Bank (PDB). Over a decade ago these proteins were divided into three structural classes, but based on the currently available wealth of structural data, we propose a new classification into six clusters, based on features of their three-dimensional structure.

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI-C 18 ) was excluded from sphingomyelinenriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholineenriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.More than 10 years ago, the hypothesis was formulated that cellular membranes are organized in discrete dynamic entities, called lipid rafts (1, 2). Studies on epithelial cell polarity revealed that lipids, in particular sphingolipids and cholesterol, were laterally organized in the exoplasmic leaflet of the apical plasma membrane according to a variable short and long range order. Furthermore, distinct proteins were shown to selectively partition into lipid rafts, indicating that rafts could serve as specific sites for molecular sorting and polarized transport.

Osmosensing and osmoregulatory compatible solute accumulation by bacteria Wood, J.M.; Bremer, Erhard; Csonka, L.N.; Kraemer, R; Poolman, B.; van der Heide, Tiemen; Smith, L.T. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. AbstractBacteria inhabit natural and artificial environments with diverse and fluctuating osmolalities, salinities and temperatures. Many maintain cytoplasmic hydration, growth and survival most effectively by accumulating kosmotropic organic Ž . Ž . solutes compatible solutes when medium osmolality is high or temperature is low above freezing . They release these solutes into their environment when the medium osmolality drops. Solutes accumulate either by synthesis or by transport from the extracellular medium. Responses to growth in high osmolality medium, including biosynthetic accumulation of trehalose, also protect Salmonella typhimurium from heat shock. Osmotically regulated transporters and mechanosensitive channels modulate cytoplasmic solute levels in Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Lactobacillus plantarum, Lactococcus lactis, Listeria monocytogenes and Salmonella typhimurium. Each organism harbours multiple osmoregulatory transporters with overlapping substrate specificities. Membrane proteins Ž that can act as both osmosensors and osmoregulatory transporters have been identified secondary transporters ProP of . E. coli and BetP of C. glutamicum as well as ABC transporter OpuA of L. lactis . The molecular bases for the modulation of gene expression and transport activity by temperature and medium osmolality are under intensive investigation with emphasis on the role of the membrane as an antenna for osmo-andror thermosensors. ᮊ 2001 Elsevier Science Inc. All rights reserved. Wood et al. r Comparati¨e Biochemistry and Physiology Part A 130 2001 437᎐460 438

The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.

Cell membranes are comprised of multicomponent lipid and protein mixtures that exhibit a complex partitioning behavior. Regions of structural and compositional heterogeneity play a major role in the sorting and self-assembly of proteins, and their clustering into higher-order oligomers. Here, we use computer simulations and optical microscopy to study the sorting of transmembrane helices into the liquid-disordered domains of phase-separated model membranes, irrespective of peptide-lipid hydrophobic mismatch. Free energy calculations show that the enthalpic contribution due to the packing of the lipids drives the lateral sorting of the helices. Hydrophobic mismatch regulates the clustering into either small dynamic or large static aggregates. These results reveal important molecular driving forces for the lateral organization and self-assembly of transmembrane helices in heterogeneous model membranes, with implications for the formation of functional protein complexes in real cells.fluorescence microscopy | lipid rafts | membrane proteins | molecular dynamics | linactants T he heterogeneity of biological membranes plays an important role in cellular function (1, 2). Despite experimental progress in recent years (3), the characterization of lateral organization in biological membranes, however, remains challenging due to the lack of tools to study fluctuating nanoscale assemblies in living cells (4-6). Model membranes (7-10) as well as isolated plasma membranes (11, 12) are more frequently studied, because largescale phase separation can be observed in these systems. In particular, at cholesterol concentrations reminiscent of biological membranes (10-30 mol % cholesterol), ternary mixtures of saturated and unsaturated lipids can segregate into coexisting lipid domains of different fluidity, a liquid-ordered (Lo) and a liquiddisordered (Ld) phase. Probing the structural and dynamical properties of these fluid domains has received a lot of attention, as it is presumably linked to the formation of lipid nanodomains ("rafts") in real cells (13,14).The structure and function of membrane proteins is intimately connected with their lipid environment (15, 16). Because of the heterogeneity of the cell membrane, proteins partition between different lipid domains, are recruited to specific locations, and form functional complexes (17)(18)(19). This lateral organization is very important for various cellular processes, such as membrane fusion (20, 21), protein trafficking (22), and signal transduction (23-25). Although lipids and integral membrane proteins are well studied by themselves, the molecular properties that determine the specific interactions between them remain poorly understood. Interactions important for protein sorting and self-assembly are (specific) protein-protein and protein-lipid forces, and indirect lipid-mediated forces. The latter category includes, for instance, forces due to entropic confinement of lipid chains and forces arising from the mismatch between the hydrophobic parts of the protein and the bi...