Improved helix and kink characterization in membrane proteins allows evaluation of kink sequence predictors

Although the α-helical secondary structure of proteins is well-defined, the exact causes and structures of helical kinks are not. This is especially important for transmembrane (TM) helices of integral membrane proteins, many of which contain kinks providing functional diversity despite predominantly helical structure. We have developed a Monte Carlo method based algorithm, MC-HELAN, to determine helical axes alongside positions and angles of helical kinks. Analysis of all nonredundant high-resolution α-helical membrane protein structures (842 TM helices from 205 polypeptide chains) revealed kinks in 64% of TM helices, demonstrating that a significantly greater proportion of TM helices are kinked than those indicated by previous analyses. The residue proline is over-represented by a factor >5 if it is two or three residues C-terminal to a bend. Prolines also cause kinks with larger kink angles than other residues. However, only 33% of TM kinks are in proximity to a proline. Machine learning techniques were used to test for sequence-based predictors of kinks. Although kinks are somewhat predicted by sequence, kink formation appears to be driven predominantly by other factors. This study provides an improved view of the prevalence and architecture of kinks in helical membrane proteins and highlights the fundamental inaccuracy of the typical topological depiction of helical membrane proteins as series of ideal helices.     

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.     

Compactization of rigid-chain amphiphilic macromolecules with local helical structure

Conformational characteristics of amphiphilic macromolecules with secondary local helical structuring are studied by the method of molecular dynamics for different properties of a helix (bending angles between neighboring vectors of the bond and internal rotation angle) and different rigidities of its fixation. Extended helices with high distances between helical turns and dense helices in which neighboring turns directly adjoin each other are studied. As the quality of a solvent deteriorates, extended helices experience a well-pronounced coil-globule transition, whose amplitude increases with an increase in chain rigidity, while the dimensions of dense helices gradually change. In a poor solvent, extended helices formed “collagen-like” structures, flexible chains of dense helices produce hairpin structures, and rigid macromolecules of dense helices form rodlike globules with an almost ideal local helical order. Independently of helix parameters, a deterioration in solvent quality leads to stabilization of the local secondary structure.     

GXXXG‐Mediated Parallel and Antiparallel Dimerization of Transmembrane Helices and Its Inhibition by Cholesterol: Single‐Pair FRET and 2D IR Studies

Small‐residue‐mediated interhelical packings are ubiquitously found in helical membrane proteins, although their interaction dynamics and lipid dependence remain mostly uncharacterized. We used a single‐pair FRET technique to examine the effect of a GXXXG motif on the association of de novo designed (AALALAA)₃ helices in liposomes. Dimerization occurred with sub‐second lifetimes, which was abolished by cholesterol. Utilizing the nearly instantaneous time‐resolution of 2D IR spectroscopy, parallel and antiparallel helix associations were identified by vibrational couplings across helices at their interface. Taken together, the data illustrate that the GXXXG motif controls helix packing but still allows for a dynamic and lipid‐regulated oligomeric state.     

Amphipathic helices as mediators of the membrane interaction of amphitropic proteins, and as modulators of bilayer physical properties

The amphipathic helix (AH) motif is used by a subset of amphitropic proteins to accomplish reversible and controlled association with the interfacial zone of membranes. Functioning as more than mere membrane anchoring domains, amphipathic helices can serve as autoinhibitory domains to suppress the protein activity in its soluble form, and as sensors or modulators of membrane curvature. Thus amphipathic helices can both respond to and modulate membrane physical properties. These and other features are illustrated by the behavior of CTP: phosphocholine cytidylyltransferase (CCT), a key regulatory enzyme in PC synthesis. A comparison of the physico-chemical features of CCT's AH motif and 10 others reveals similarities and several differences. The importance of these parameters to the particulars of the membrane interaction and to functional consequences requires more systematic exploration. The membrane partitioning of amphitropic proteins with AH motifs can be regulated by various strategies including changes in membrane lipid composition, phosphorylation, ligand-induced conformational changes, and membrane curvature. Several amphitropic proteins that control budding or tubule formation in cells have AH motifs. The insertion of the hydrophobic face of these amphipathic helices generates an asymmetry in the lateral pressure of the two leaflets resulting in an induction of positive curvature. Curvature induction or stabilization may be a universal property of AHA proteins, not just those involved in budding, but this possibility requires further demonstration.     

Development of 7TM receptor-ligand complex models using ligand-biased, semi-empirical helix-bundle repacking in torsion space: application to the agonist interaction of the human dopamine D2 receptor

Prediction of 3D structures of membrane proteins, and of G-protein coupled receptors (GPCRs) in particular, is motivated by their importance in biological systems and the difficulties associated with experimental structure determination. In the present study, a novel method for the prediction of 3D structures of the membrane-embedded region of helical membrane proteins is presented. A large pool of candidate models are produced by repacking of the helices of a homology model using Monte Carlo sampling in torsion space, followed by ranking based on their geometric and ligand-binding properties. The trajectory is directed by weak initial restraints to orient helices towards the original model to improve computation efficiency, and by a ligand to guide the receptor towards a chosen conformational state. The method was validated by construction of the β₁ adrenergic receptor model in complex with (S)-cyanopindolol using bovine rhodopsin as template. In addition, models of the dopamine D₂ receptor were produced with the selective and rigid agonist (R)-N-propylapomorphine ((R)-NPA) present. A second quality assessment was implemented by evaluating the results from docking of a library of 29 ligands with known activity, which further discriminated between receptor models. Agonist binding and recognition by the dopamine D₂ receptor is interpreted using the 3D structure model resulting from the approach. This method has a potential for modeling of all types of helical transmembrane proteins for which a structural template with sequence homology sufficient for homology modeling is not available or is in an incorrect conformational state, but for which sufficient empirical information is accessible.     

Detergent-associated solution conformations of helical and beta-barrel membrane proteins

Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the beta-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.     

On the origin of helical mesostructures

The investigation on the formation mechanism of helical structures and the synthesis of helical materials is attractive for scientists in different fields. Here we report the synthesis of helical mesoporous materials with chiral channels in the presence of achiral surfactants. More importantly, we suggest a simple and purely interfacial interaction mechanism to explain the spontaneous formation of helical mesostructures. Unlike the proposed model for the formation of helical molecular chains or surpramolecular packing based on the geometrically motivated model or the entropically driven model, the origin of the helical mesostructured materials may be attributed to a morphological transformation accompanied by a reduction in surface free energy. After the helical morphology is formed, the increase in bending energy together with the derivation from a perfect hexagonal mesostructure may limit the curvature of helices. Our model may be general and important in the designed synthesis of helical mesoporous materials.     

Self‐assembly of supramolecular polymers into tunable helical structures

There is growing interest in the design of synthetic molecules that are able to self‐assemble into a polymeric chain with compact helical conformations, which is analogous to the folded state of natural proteins. Herein, we highlight supramolecular approach to the formation of helical architectures and their conformational changes driven by external stimuli. Helical organization in synthetic self‐assembling systems can be achieved by the various types of noncovalent interactions, which include hydrogen bonding, solvophobic effects, and metal‐ligand interactions. Since the external environment can have a large influence on the strength and configuration of noncovalent interactions between the individual components, stimulus‐induced alterations in the intramolecular noncovalent interactions can result in dynamic conformational change of the supramolecular helical structure thus, driving significant changes in the properties of the materials. Therefore, these supramolecular helices hold great promise as stimuli‐responsive materials. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 1925–1935, 2008     

Multidimensional umbrella sampling and replica‐exchange molecular dynamics simulations for structure prediction of transmembrane helix dimers

Structural information of a transmembrane (TM) helix dimer is useful in understanding molecular mechanisms of important biological phenomena such as signal transduction across the cell membrane. Here, we describe an umbrella sampling (US) scheme for predicting the structure of a TM helix dimer in implicit membrane using the interhelical crossing angle and the TM–TM relative rotation angles as the reaction coordinates. This scheme conducts an efficient conformational search on TM–TM contact interfaces, and its robustness is tested by predicting the structures of glycophorin A (GpA) and receptor tyrosine kinase EphA1 (EphA1) TM dimers. The nuclear magnetic resonance (NMR) structures of both proteins correspond to the global free‐energy minimum states in their free‐energy landscapes. In addition, using the landscape of GpA as a reference, we also examine the protocols of temperature replica‐exchange molecular dynamics (REMD) simulations for structure prediction of TM helix dimers in implicit membrane. A wide temperature range in REMD simulations, for example, 250–1000 K, is required to efficiently obtain a free‐energy landscape consistent with the US simulations. The interhelical crossing angle and the TM–TM relative rotation angles can be used as reaction coordinates in multidimensional US and be good measures for conformational sampling of REMD simulations. © 2013 Wiley Periodicals, Inc.     

Deuterium N.M.R. studies of polypeptides I. Sidechain orientation in poly(γ-benzyl L-glutamate) and the mechanism of the cholesteric sense inversion

The mechanism of thermally- and solvent-induced cholesteric sense inversions in lyotropic polypeptide liquid crystals has been discussed based on deuterium N.M.R. observations for poly(γ-benzyl L-glutamate) with perdeuteriated sidechain benzyl ester groups. Comparison of the deuterium quadrupolar splitting pattern with the macroscopic helical twisting power indicates that the sense inversion does not necessarily require sidechain conformational transitions (or changes in the helix surface chirality). The new data support a less specific mechanism for sense determination in polypeptide liquid crystals: anisotropic intermolecular interactions between helices are influenced by the solvent dielectric medium.     

String kernels and high-quality data set for improved prediction of kinked helices in α-helical membrane proteins

The reasons for distortions from optimal α-helical geometry are widely unknown, but their influences on structural changes of proteins are significant. Hence, their prediction is a crucial problem in structural bioinformatics. For the particular case of kink prediction, we generated a data set of 132 membrane proteins containing 1014 manually labeled helices and examined the environment of kinks. Our sequence analysis confirms the great relevance of proline and reveals disproportionately high occurrences of glycine and serine at kink positions. The structural analysis shows significantly different solvent accessible surface area mean values for kinked and nonkinked helices. More important, we used this data set to validate string kernels for support vector machines as a new kink prediction method. Applying the new predictor, about 80% of all helices could be correctly predicted as kinked or nonkinked even when focusing on small helical fragments. The results exceed recently reported accuracies of alternative approaches and are a consequence of both the method and the data set.     

Shear-induced conformational ordering, relaxation, and crystallization of isotactic polypropylene

The shear-induced coil-helix transition of isotactic polypropylene (iPP) has been studied with time-resolved Fourier transform infrared spectroscopy at various temperatures. The effects of temperature, shear rate, and strain on the coil-helix transition were studied systematically. The induced conformational order increases with the shear rate and strain. A threshold of shear strain is required to induce conformational ordering. High temperature reduces the effect of shear on the conformational order, though a simple correlation was not found. Following the shear-induced conformational ordering, relaxation of helices occurs, which follows the first-order exponential decay at temperatures well above the normal melting point of iPP. The relaxation time versus temperature is fitted with an Arrhenius law, which generates an activation energy of 135 kJ/mol for the helix-coil transition of iPP. At temperatures around the normal melting point, two exponential decays are needed to fit well on the relaxation kinetic of helices. This suggests that two different states of helices are induced by shear: (i) isolated single helices far away from each other without interactions, which have a fast relaxation kinetic; (ii) aggregations of helices or helical bundles with strong interactions among each other, which have a much slower relaxation process. The helical bundles are assumed to be the precursors of nuclei for crystallization. The different helix concentrations and distributions are the origin of the three different processes of crystallization after shear. The correlation between the shear-induced conformational order and crystallization is discussed.     

Modulation of the spontaneous curvature and bending rigidity of lipid membranes by interfacially adsorbed amphipathic peptides

Amphipathic alpha-helical peptides are often ascribed an ability to induce curvature stress in lipid membranes. This may lead directly to a bending deformation of the host membrane, or it may promote the formation of defects that involve highly curved lipid layers present in membrane pores, fusion intermediates, and solubilized peptide-micelle complexes. The driving force is the same in all cases: peptides induce a spontaneous curvature in the host lipid layer, the sign of which depends sensitively on the peptide's structural properties. We provide a quantitative account for this observation on the basis of a molecular-level method. To this end, we consider a lipid membrane with peptides interfacially adsorbed onto one leaflet at high peptide-to-lipid ratio. The peptides are modeled generically as rigid cylinders that interact with the host membrane through a perturbation of the conformational properties of the lipid chains. Through the use of a molecular-level chain packing theory, we calculate the elastic properties, that is, the spontaneous curvature and bending stiffness, of the peptide-decorated lipid membrane as a function of the peptide's insertion depth. We find a positive spontaneous curvature (preferred bending of the membrane away from the peptide) for small penetration depths of the peptide. At a penetration depth roughly equal to half-insertion into the hydrocarbon core, the spontaneous curvature changes sign, implying negative spontaneous curvature (preferred bending of the membrane toward the peptide) for large penetration depths. Despite thinning of the membrane upon peptide insertion, we find an increase in the bending stiffness. We discuss these findings in terms of how the peptide induces elastic stress.     

Transient Helicity: Fuel‐Driven Temporal Control over Conformational Switching in a Supramolecular Polymer

Synthetic manifestations of supramolecular chirality have extensively drawn inspiration from naturally occurring systems. Even though in biological systems conformational changes are dissipative, synthetic systems that change conformation under non‐equilibrium conditions have still not been established. Herein, we attempt to alleviate this scenario by reporting a synthetic supramolecular system that undergoes temporal changes in its helical conformation as an active system at the expense of a biologically relevant chemical fuel, ATP. Use of enzymes working in tandem provides transient and switchable helices with modular lifetime and stereomutation rates.     

Probing possible left- and right-handed poly(dinucleotide) helical conformations from (n–h) plots. models for polysequential nucleotides

Introducing the concept of the “dinucleotide” as the helical repeat, theoretical attempts have been made to determine possible single and double stranded helical structures by using helical parameter calculations and model building investigations. By virtue of its flexible framework, the dinucleotide repeat offers a much greater scope of finding new secondary structural forms for nucleic acids. Considering only those conformations which show tendency for at least partial base overlap as does the dinucleotide helical repeat, it has been possible to predict poly(dinucleotide) helical models in which successive phosphodiesters as well as nucleotide conformations alternate. More important, the recently found left-handed Z-type polynucleotide helix is characterized rather uniquely on the helical parameter plot. The results further suggest the possibility of other Z-type helices obtainable by alternative conformations for the exocyclic C4'–C5' bond and sugar pucker. Near neighbor long range conformational correlations between the dinucleotide repeat and the phosphodiester linking them have been established similar to poly(mononucleotide) helices. Need for considering higher repeats such as trinucleotide has been suggested to obtain models for looped out helical conformations.     

Heterocyclic peptide backbone modifications in an alpha-helical coiled coil

In this paper, we present 1,2,3-triazole epsilon2-amino acids incorporated as a dipeptide surrogate at three positions in the sequence of a known alpha-helical coiled coil. Biophysical characterization indicates that the modified peptides retain much of the helical structure of the parent sequence, and that the thermodynamic stability of the coiled coil depends on the position of the incorporation of the epsilon-residue. Crystal structures obtained for each peptide give insight into the chemical behavior and conformational preferences of the non-natural amino acid and show that the triazole ring can participate in the backbone hydrogen bonding of the alpha-helix as well as template an interhelical crossing between chains in the bundle.     

Magic angle spinning and oriented sample solid-state NMR structural restraints combine for influenza a M2 protein functional insights

As a small tetrameric helical membrane protein, the M2 proton channel structure is highly sensitive to its environment. As a result, structural data from a lipid bilayer environment have proven to be essential for describing the conductance mechanism. While oriented sample solid-state NMR has provided a high-resolution backbone structure in lipid bilayers, quaternary packing of the helices and many of the side-chain conformations have been poorly restrained. Furthermore, the quaternary structural stability has remained a mystery. Here, the isotropic chemical shift data and interhelical cross peaks from magic angle spinning solid-state NMR of a liposomal preparation strongly support the quaternary structure of the transmembrane helical bundle as a dimer-of-dimers structure. The data also explain how the tetrameric stability is enhanced once two charges are absorbed by the His37 tetrad prior to activation of this proton channel. The combination of these two solid-state NMR techniques appears to be a powerful approach for characterizing helical membrane protein structure.     

Dynamic Axial-to-Helical Communication Mechanism in Poly[(allenylethynylenephenylene)acetylene]s under External Stimuli

Helix inversion in chiral dynamic helical polymers is usually achieved by conformational changes at the pendant groups induced through external stimuli. Herein, a different mechanism of helix inversion in poly(phenylacetylene)s (PPAs) is presented, based on the activation/deactivation of supramolecular interactions. We prepared poly[(allenylethynylenephenylene)acetylene]s (PAEPAs) in which the pendant groups are conformationally locked chiral allenes. Therefore, their substituents are placed in specific spatial orientations. As a result, the screw sense of a PAEPA is fixed by the allenyl substituent with the optimal size/distance relationship to the backbone. This helical sense command can be surpassed by supramolecular interactions between another substituent on the allene and appropriate external stimuli, such as amines. So, a helix inversion occurs through a novel axial-to-helical communication mechanism, opening a new scenario for taming the helices of chiral dynamic helical polymers.