Shape transitions in lipid membranes and protein mediated vesicle fusion and fission

In the cell, the plasma membrane is often densely decorated by transmembrane proteins. The morphology and dynamics of the membrane are strongly influenced by the presence of proteins. In this paper, we use a coarse-grained model to explore the composite membrane-protein system and develop a simulation methodology based on thermodynamic integration to examine free energy changes during membrane shape transitions. The authors show that a critical concentration of conical membrane proteins or proteins with nonzero spontaneous curvature can drive the formation of small vesicles. The driving force of vesicle budding stems from the preference of proteins to gather in regions of high curvature. A sufficiently high concentration of proteins therefore can influence the topology of the membrane. The biological significance of our results is discussed.     

Crystalline protein domains and lipid bilayer vesicle shape transformations

Cellular membranes can take on a variety of shapes to assist biological processes including endocytosis. Membrane-associated protein domains provide a possible mechanism for determining membrane curvature. We study the effect of tethered streptavidin protein crystals on the curvature of giant unilamellar vesicles (GUVs) using confocal, fluorescence, and differential interference contrast microscopy. Above a critical protein concentration, streptavidin domains align and percolate as they form, deforming GUVs into prolate spheroidal shapes in a size-dependent fashion. We propose a mechanism for this shape transformation based on domain growth and jamming. Osmotic deflation of streptavidin-coated GUVs reveals that the relatively rigid streptavidin protein domains resist membrane bending. Moreover, in contrast to highly curved protein domains that facilitate membrane budding, the relatively flat streptavidin domains prevent membrane budding under high osmotic stress. Thus, crystalline streptavidin domains are shown to have a stabilizing effect on lipid membranes. Our study gives insight into the mechanism for protein-mediated stabilization of cellular membranes.     

Intrinsically cell-permeable miniature proteins based on a minimal cationic PPII motif

Cell-penetrating peptides (CPPs) provide promising tools for the cellular delivery of molecular cargos ranging in size from small molecules and peptides to proteins and quantum dots. CPPs are typically cationic and/or amphipathic sequences that are unstructured or alpha-helical. We expand the repertoire of cell-penetrating motifs by designing encodable CPPs possessing type-II polyproline (PPII) helical structure. These motifs surpass the uptake efficiency of existing CPPs and are not cytotoxic at concentrations 100 times greater than that necessary for delivery. By replacing the PPII helix of a miniature protein, the motif can endow intrinsic cell permeability without increasing molecular size.     

Membrane-dependent effects of a cytoplasmic helix on the structure and drug binding of the influenza virus M2 protein

The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22-46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45-62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21-61). (13)C-(2)H distance measurements of (13)C-labeled protein and (2)H-labeled amantadine showed that in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers, the first equivalent of drug bound S31 inside the M2(21-61) pore, similar to the behavior of M2 transmembrane peptide (M2TM) in DMPC bilayers. The nonspecific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21-61) is S31 in the TM pore. Interestingly, when M2(21-61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, whereas M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state but did not rescue drug binding to M2(21-61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helix to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein.     

Basic charge clusters and predictions of membrane protein topology

The topology predictor SPLIT 4.0 (http://pref.etfos.hr) predicts the sequence location of transmembrane helices by performing an automatic selection of optimal amino acid attribute and corresponding preference functions. The best topological model is selected by choosing the highest absolute bias parameter that combines the bias in basic charge motifs and the bias in positive residues (the "positive inside rule") with the charge difference across the first transmembrane segment. Basic charge motifs, such as the BBB, BXXBB, and BBXXB motifs in alpha-helical integral membrane proteins, are significantly more frequent near cytoplasmic membrane surface than expected from the Arg/Lys (B) frequency. The predictor's accuracy is 99% for predicting 178 transmembrane helices in all membrane proteins or subunits of known 3D structure.     

Dipolar waves map the structure and topology of helices in membrane proteins

Dipolar waves describe the structure and topology of helices in membrane proteins. The fit of sinusoids with the 3.6 residues per turn period of ideal alpha-helices to experimental measurements of dipolar couplings as a function of residue number makes it possible to simultaneously identify the residues in the helices, detect kinks or curvature in the helices, and determine the absolute rotations and orientations of helices in completely aligned bilayer samples and relative rotations and orientations of helices in a common molecular frame in weakly aligned micelle samples. Since as much as 80% of the structured residues in a membrane protein are in helices, the analysis of dipolar waves provides a significant step toward structure determination of helical membrane proteins by NMR spectroscopy.     

Membrane domain-disrupting effects of 4-substitued cholesterol derivatives

A wide range of cellular functions are thought to be regulated not only by the activity of membrane proteins, but also by the local membrane organization, including domains of specific lipid composition. Thus, molecules and drugs targeting and disrupting this lipid pattern, particularly of the plasma membrane, will not only help to investigate the role of membrane domains in cell biology, but might also be interesting candidates for therapy. We have identified three 4-substituted cholesterol derivatives that are able to induce a domain-disrupting effect in model membranes. When applied to giant unilamellar vesicles displaying liquid-ordered-liquid-disordered phase coexistence, extensive reorganization of the membrane can be observed, such as the budding of membrane tubules or changes in the geometry of the domains, to the point of complete abolition of phase separation. In this case, the resulting membranes display a fluidity intermediate between those of liquid-disordered and liquid-ordered phases.     

Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

Positive reinforcement for viruses

Virus-cell membrane fusion requires a critical transition from positive to negative membrane curvature. St.?Vincent et?al. (2010), in PNAS, designed a class of antivirals that targets this transition. These rigid amphipathic fusion inhibitors are active against an array of enveloped viruses.     

Novel features for identifying A-minors in three-dimensional RNA molecules

RNA tertiary interactions or tertiary motifs are conserved structural patterns formed by pairwise interactions between nucleotides. They include base-pairing, base-stacking, and base-phosphate interactions. A-minor motifs are the most common tertiary interactions in the large ribosomal subunit. The A-minor motif is a nucleotide triple in which minor groove edges of an adenine base are inserted into the minor groove of neighboring helices, leading to interaction with a stabilizing base pair. We propose here novel features for identifying and predicting A-minor motifs in a given three-dimensional RNA molecule. By utilizing the features together with machine learning algorithms including random forests and support vector machines, we show experimentally that our approach is capable of predicting A-minor motifs in the given RNA molecule effectively, demonstrating the usefulness of the proposed approach. The techniques developed from this work will be useful for molecular biologists and biochemists to analyze RNA tertiary motifs, specifically A-minor interactions.     

β-Peptides as Inhibitors of Small-Intestinal Cholesterol and Fat Absorption

Selective lipid transport through the brush-border membrane in the small intestine of mammals is mediated by membrane-bound proteins, the so-called scavenger receptors of class B, type I or II (SR-BI or -BII). These, in turn, are inhibited by certain proteins and synthetic α-peptides that have an amphipathic helix as the binding motif (Fig. 1). In whole cells (test with human colonic carcinoma cells, CaCo-2), on the other hand, the inhibitors are subject to proteolysis. We have now tested six β-peptides (hexa-, hepta-, and nonamers 1 – 6 ), each carrying one to seven water-solubilizing side chains of either Ser or Lys, with a brush-border-membrane (BBM) vesicle model system (rate and IC₅₀ values in Figs. 2 and 3) and with a tightly packed monolayer of CaCo-2 cells (rate in Fig. 4), to find that the rate of transport of cholesterol can be reduced to what may be considered the passive diffusion (`background') level. There is a correlation between the ability of the β-peptides to form an amphipathic-type 3₁₄-helical secondary structure in MeOH and their inhibitory effect (Table 1 and Fig. 5). Although the inhibitory activity of the β-peptides is in only the mM range (Table 2), it is to be compared with no activity at all of previously tested α-peptides and proteins (built of L -amino acids) in CaCo-2 cells. Furthermore, these active β-peptides ( 1 , 5 , and 6 ) contain only seven or nine residues and must be considered simple, first-generation models capable of mimicking the biological activity of amphipathic α-peptide helices in living whole cells.     

Effect of Phosphorylation on the Structural Behaviour of Peptides Derived from the Intrinsically Disordered C-Terminal Domain of Histone H1.0

To investigate the structural impact of phosphorylation on the human histone H1.0 C-terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T¹¹⁸-H1.0 (T¹¹⁸PKK motif) and T¹⁴⁰-H1.0 (T¹⁴⁰PVK motif). Both model peptides are mainly disordered in aqueous solution in their non-phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T¹¹⁸-H1.0 and pT¹¹⁸-H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104–115 and a short α/3₁₀ helix (residues 119–123), that are almost perpendicular in T¹¹⁸-H1.0 but have a poorly defined orientation in pT¹¹⁸-H1.0. Peptides T¹⁴⁰-H1.0 and pT¹⁴⁰-H1.0 form very similar α helices between residues 141–147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side-chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT¹¹⁸-H1.0 and pT¹⁴⁰-H1.0 has pK_a values below the intrinsic values, which can be explained by non-specific charge–charge interactions with nearby Lys. The non-polar Val in the TPVK motif accounts for the pT¹⁴⁰ pK_a being closer to the intrinsic pK_a value than the pT¹¹⁸ pK_a. Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short-lived intrinsically disordered proteins and domains.     

The influence of anisotropic membrane inclusions on curvature elastic properties of lipid membranes

A membrane inclusion can be defined as a complex of protein or peptide and the surrounding significantly distorted lipids. We suggest a theoretical model that allows for the estimation of the influence of membrane inclusions on the curvature elastic properties of lipid membranes. Our treatment includes anisotropic inclusions whose energetics depends on their in-plane orientation within the membrane. On the basis of continuum elasticity theory, we calculate the inclusion-membrane interaction energy that reflects the protein or peptide-induced short-ranged elastic deformation of a bent lipid layer. A numerical estimate of the corresponding interaction constants indicates the ability of inclusions to sense membrane bending and to accumulate at regions of favorable curvature, matching the effective shape of the inclusions. Strongly anisotropic inclusions interact favorably with lipid layers that adopt saddlelike curvature; such structures may be stabilized energetically. We explore this possibility for the case of vesicle budding where we consider a shape sequence of closed, axisymmetric vesicles that form a (saddle-curvature adopting) membrane neck. It appears that not only isotropic but also strongly anisotropic inclusions can significantly contribute to the budding energetics, a finding that we discuss in terms of recent experiments.     

Molecular models of lipopeptide detergents: large coiled-coils with hydrocarbon interiors

We have constructed molecular models of octameric micelles formed by a recently developed lipopeptide detergent consisting of a single amphipathic alpha-helix coupled to two acyl chains at either end of the helix. The models explain the experimentally observed aggregation behavior of peptides with different acyl chain lengths. The octameric micelles form a unique coiled-coil structure, with the acyl chains in a nearly frozen conformation inside the cylindrical assemblies. Two extreme models with helices either all parallel or in an alternating orientation suggest that the alternating orientation is energetically more favorable. The models suggest several new directions for further diversifying this new class of detergents for the structural studies of membrane proteins.     

Introduction of curvature in amphipathic oligothiophenes for defined aggregate formation

In this study the possibility to control the size and shape of self-assembled structures through the local curvature of their molecular building blocks has been investigated. To this end a series of amphipathic conjugated oligothiophenes with a well-defined curvature of their backbone has been designed and synthesized. The molecular (local) curvature of these oligothiophenes resulted from a preference for cis instead of trans conformations at specific positions along the oligothiophene backbone, which can be controlled by the sequence of hydrophilic and hydrophobic groups, while their ratio was kept constant. The self-assembly of ter-, sexi-, and dodecathiophenes appeared to be a low-cooperative process, involving the formation of premicellar aggregates at sub-millimolar concentrations, which at concentrations in the millimolar regime transformed into micelles and cylindrical micelles. The aggregates display fine structures with dimensions reminiscent of the thiophene molecules. The structure-morphology relationship of the ter- and sexithiophenes could be described by conventional packing theory. However, with the dodecathiophene, the backbone curvature governed the formation of cylindrical aggregates with a well-defined diameter. These results demonstrate that it is possible to control the aggregation morphology of simple amphipathic oligothiophenes by implementation of an additional structural motif namely, the curvature.     

Curvature sorting of peripheral proteins on solid-supported wavy membranes

Cellular membrane deformation and the associated redistribution of membrane-bound proteins are important aspects of membrane function. Current model membrane approaches for studying curvature sensing are limited to positive curvatures and often require complex and delicate experimental setups. To overcome these challenges, we fabricated a wavy substrate by imposing a range of curvatures onto an adhering lipid bilayer membrane. We examined the curvature sorting of several peripheral proteins binding to the wavy membrane and observed them to partition into distinct regions of curvature. Furthermore, single-molecule imaging experiments suggested that the curvature sensing of proteins on low-curvature substrates requires cooperative interactions.     

Parallel Homochiral and Anti‐Parallel Heterochiral Hydrogen‐Bonding Interfaces in Multi‐Helical Abiotic Foldamers

A hydrogen‐bonding interface between helical aromatic oligoamide foldamers has been designed to promote the folding of a helix‐turn‐helix motif with a head‐to‐tail arrangement of two helices of opposite handedness. This design complements an earlier helix‐turn‐helix motif with a head‐to‐head arrangement of two helices of identical handedness interface. The two motifs were shown to have comparable stability and were combined in a unimolecular tetra‐helix fold constituting the largest abiotic tertiary structure to date.