Artificial beta-sheets: chemical models of beta-sheets

Chemical models provide tools with which to simplify and study complicated biological systems. Forces and chemical processes that govern the structure, function, and interactions of a biomacromolecule can be explored with a simple, easy-to-study synthetic molecule. Chemical models of beta-sheet structures have helped to elucidate the factors influencing protein structures and functions. Chemical models that mimic beta-sheet quaternary structure and interactions are emerging as valuable tools with which to better understand and control protein recognition and protein aggregation.     

Bending undulations and elasticity of the erythrocyte membrane: effects of cell shape and membrane organization

The undulatory excitations (flickering) of human and camel erythrocytes were evaluated by employing the previously used flicker spectroscopy and by local measurements of the autocorrelation function K (t) of the cell thickness fluctuations using a dynamic image processing technique. By fitting theoretical and experimental flicker spectra relative values of the bending elastic modulus K _c of the membrane and of the cytoplasmic viscosity were obtained. The effects of shape changes were monitored by simultaneous measurement of the average light intensity I ₀ passing the cells and by phase contrast microscopic observation of the cells. Evaluation of the cellular excitations in terms of the quasi-spherical model yielded values of K _c /R _inf0 ^sup3 and · R ₀ (R ₀=equivalent sphere radius) and allowed us to account (1) for volume changes, (2) for effects of surface tension and spontaneous curvature and (3) for the non-exponential decay of K (t). From the long time decay of K (t) we obtained an upper limit of the bending elastic modulus of normal cells of K _c = 2–3 · 10^–19 Nm which is an order of magnitude larger than the value found by reflection interference contrast microscopy (RICT, K _c , = 3.4 · 10^–20 Nm, Zilker et al. 1987) but considerably lower than expected for a bilayer containing 50% cholesterol (K _c = 5 · 10^–19 Nm, Duwe et al. 1989). The major part of the paper deals with long time measurements (order of hours) of variations of the apparent K _c and values of single cells (and their reversibility) caused (1) by osmotic volume changes, (2) by discocytestomatocyte transitions induced by albumin and triflouperazine, (3) by discocyte-echinocyte transitions induced by expansion of the lipid/protein bilayer (by incubation with lipid vesicles) and by ATP-depletion in physiological NaCI solution, (4), by coupling or decoupling of bilayer and cytoskeleton using wheat germ agglutinin or erythrocytes with elliptocytosis and (5) by cross-linking the cytoskeleton using diamide. These experiments showed: (1) K _c and are minimal at physiological osmolarity and temperature and well controlled over a large range of these parameters. (2) Echinocyte formation does not markedly alter the apparent membrane bending stiffness. (3) During swelling the cell may undergo a transient discocyte-stomatocyte transition. (4) Strong increases of the apparent K _c and after cup-formation or strong swelling and deflation are due to the effect of shear elasticity and surface tension. Our major conclusions are: (1) The erythrocyte membrane exhibits a shear free deformation regime which requires ATP for its maintenance. (2) Shape transitions may be caused by relative area changes either of the two monolayers of the lipid/protein bilayer (corresponding to the bilayer coupling hypothesis) or of the bilayer and the cytoskeleton where the latter mechanism appears to be more frequent. (3) The low bending stiffness and the shear free deformation regime are explained in terms of a slight excess area of the lipid bilayer leading to a pre-undulated surface profile. Freeze fracture electron microscopy studies provide direct evidence for a pre-undulated bilayer with an undulation wavelength of approximately 100 nm. Offprint requests to: E. Sackmann     

Infrared dichroism of isotope-edited alpha-helices and beta-sheets

Isotope editing of amide infrared bands not only localises secondary structural elements within the protein but also yields conformational information that is not available from the linear dichroism of aligned samples without isotope editing. The additional information that can be derived on the orientational distribution of alpha-helices in membranes by the combined use of different amide bands and several positions of labelling is presented here. Also, the relationship between the azimuthal orientation of the transition moment and the protein structure is treated explicitly. A comprehensive analysis of the infrared dichroism for beta-sheets and beta-barrels is given here, for the first time. The orientation of the individual transition moments in a beta-sheet that is essential for this analysis is derived for the different amide bands.     

Twist and shear in beta-sheets and beta-ribbons

The structures of the beta-sheets and the beta-ribbons have been analysed using high-resolution protein structure data. Systematic asymmetries measured in both parallel and antiparallel beta-structures include the sheet twist and the strand shear. In order to determine the origin of these asymmetries, numerous interactions and correlations were examined. The strongest correlations are observed for residues in antiparallel beta-sheets and beta-ribbons that form non-H-bonded pairs. For these residues, the sheet twist is correlated to the backbone phi angle but not to the psi angle. Our analysis supports the existence of an inter-strand C(alpha)H(alpha)...O weak H-bond, which, together with the CO...HN H-bond, constitutes a bifurcated H-bond that links neighbouring beta-strands. Residues of beta-sheets and beta-ribbons in high-resolution protein structures form a distinct region of the Ramachandran plot, which is determined by the formation of the bifurcated H-bond, the formation of an intra-strand O...H(alpha) non-bonded polar interaction, and an intra-strand O...C(beta) steric clash. Using beta-strands parameterised by phi-psi values from the allowed beta-sheet region of the Ramachandran plot, the shear and the right-hand twist can be reproduced in a simple model of the antiparallel and parallel beta-ribbon that models the bifurcated H-bonds specifically. The conformations of interior residues of beta-sheets are shown to be subsets of the conformations of residues of beta-ribbons.     

Reversible unfolding of beta-sheets in membranes: a calorimetric study

The hexapeptide acetyl-Trp-Leu(5) (AcWL(5)) has the remarkable ability to assemble reversibly and spontaneously into beta-sheets on lipid membranes as a result of monomer partitioning followed by cooperative assembly. This system provides a unique opportunity to study the thermodynamics of protein folding in membranes, which we have done using isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). The results, which may represent the first example of reversible thermal unfolding of peptides in membranes, help to define the contribution of hydrogen bonding to the extreme thermal stability of membrane proteins. ITC revealed that the enthalpy change for partitioning of monomeric, unstructured AcWL(5) from water into membranes was zero within experimental error over the temperature range of 5 degrees C to 75 degrees C. DSC showed that the beta-sheet aggregates underwent a reversible, endothermic, and very asymmetric thermal transition with a concentration-dependent transition temperature (T(m)) in the range of 60 degrees C to 80 degrees C. A numerical model of nucleation and growth-dependent assembly of oligomeric beta-sheets, proposed earlier to describe beta-sheet formation in membranes, recreated remarkably well the unusual shape and concentration-dependence of the transition peaks. The enthalpy for thermal unfolding of AcWL(5) beta-sheets in the membrane was found to be about 8(+/-1)kcal mol(-1), or about 1.3(+/-0.2)kcal mol(-1) per residue.     

Analysis and prediction of inter-strand packing distances between beta-sheets of globular proteins

Any two beta-strands belonging to two different beta-sheets in a protein structure are considered to pack interactively if each beta-strand has at least one residue that undergoes a loss of one tenth or more of its solvent contact surface area upon packing. A data set of protein 3-D structures (determined at 2.5 A resolution or better), corresponding to 428 protein chains, contains 1986 non-identical pairs of beta-strands involved in interactive packing. The inter-axial distance between these is significantly correlated to the weighted sum of the volumes of the interacting residues at the packing interface. This correlation can be used to predict the changes in the inter-sheet distances in equivalent beta-sheets in homologous proteins and, therefore, is of value in comparative modelling of proteins.     

Amino acid pairing preferences in parallel beta-sheets in proteins

Statistical approaches have been applied to examine amino acid pairing preferences within parallel beta-sheets. The main chain hydrogen bonding pattern in parallel beta-sheets means that, for each residue pair, only one of the residues is involved in main chain hydrogen bonding with the strand containing the partner residue. We call this the hydrogen bonded (HB) residue and the partner residue the non-hydrogen bonded (nHB) residue, and differentiate between the favorability of a pair and that of its reverse pair, e.g. Asn(HB)-Thr(nHB)versus Thr(HB)-Asn(nHB). Significantly (p < or = 0.000001) favoured pairings were rationalised using stereochemical arguments. For instance, Asn(HB)-Thr(nHB) and Arg(HB)-Thr(nHB) were favoured pairs, where the residues adopted favoured chi1 rotamer positions that allowed side-chain interactions to occur. In contrast, Thr(HB)-Asn(nHB) and Thr(HB)-Arg(nHB) were not significantly favoured, and could only form side-chain interactions if the residues involved adopted less favourable chi1 conformations. The favourability of hydrophobic pairs e.g. Ile(HB)-Ile(nHB), Val(HB)-Val(nHB) and Leu(HB)-Ile(nHB) was explained by the residues adopting their most preferred chi1 and chi2 conformations, which enabled them to form nested arrangements. Cysteine-cysteine pairs are significantly favoured, although these do not form intrasheet disulphide bridges. Interactions between positively and negatively charged residues were asymmetrically preferred: those with the negatively charged residue at the HB position were more favoured. This trend was accounted for by the presence of general electrostatic interactions, which, based on analysis of distances between charged atoms, were likely to be stronger when the negatively charged residue is the HB partner. The Arg(HB)-Asp(nHB) interaction was an exception to this trend and its favorability was rationalised by the formation of specific side-chain interactions. This research provides rules that could be applied to protein structure prediction, comparative modelling and protein engineering and design. The methods used to analyse the pairing preferences are automated and detailed results are available (http://www.rubic.rdg.ac.uk/betapairprefsparallel/).     

Context-dependence of amino acid residue pairing in antiparallel beta-sheets.

In an effort to understand the driving forces behind antiparallel beta-sheet assembly, we have investigated the mutational tolerance of four pairs of residues in CspA, the major cold shock protein of E. coli. Two buried pairs and two exposed pairs of neighboring amino acids were separately randomized and the corresponding effects on protein stability were assessed using a protein expression screen. The thermal denaturation of a subset of the recovered proteins was measured by circular dichroism spectroscopy in order to determine the range of stabilities sampled by the expressed mutants. As anticipated, buried sites are substantially less tolerant of substitutions than exposed sites with more than half of the exposed residue combinations giving rise to stably folded proteins. The two exposed residue pairs, however, display different degrees of tolerance to substitution and accept different residue pair combinations. Except for the prohibition of proline from interior strand positions, no obvious correlations of mutant stability with any single parameter such as beta-sheet propensity or hydrophobicity can be detected. Mutant combinations recovered in both orientations (e.g. XY and YX) at a given exposed pair site often show markedly different stabilities, indicating that the local environment plays a substantial role in modulating the pairing preferences of residues in beta-sheets.     

Theoretical determination of the CD of proteins containing closely packed antiparallel beta-sheets

Proteins containing two closely packed β-sheets comprise an important class of biopolymers. Rotational and dipole strengths have been determined by the excition coupling model for interacting pairs of two idealized flat β-sheets and for the double β-sheets of seven globular proteins: plastocyanin, human prealbumin, immunoglobulin V_REI, concanavalin A, Cu,Zn superoxide dismutase, staphylococcal nuclease, and elastase. The effects of various geometrical factors on the CD spectra were investigated. Results for the idealized sheets indicate that two sheets display through-space interactions that are large at distances of 5–7 Å and remain significant even at distances typical of the intersheet separations in globular proteins (12–15 Å). The CD spectra are sensitive to the angle (Ω) between the strand directions of the two sheets, with maximum intersheet contributions at Ω = ±45°. Both the intrastrand and interstrand twisting were determined in the seven proteins, and their effects on the calculated CD are discussed. This work represents the first theoretical CD study on the interactions of two regular protein secondary structures, including rotational strength calculations on large sections (up to 135 residues) of globular proteins.     

Natural polypeptide scaffolds: beta-sheets, beta-turns, and beta-hairpins

This paper provides an introduction to fundamental conformational states of polypeptides in the beta-region of phi,psi space, in which the backbone is extended near to its maximal length, and to more complex architectures in which extended segments are linked by turns and loops. There are several variants on these conformations, and they comprise versatile scaffolds for presentation of side chains and backbone amides for molecular recognition and designed catalysts. In addition, the geometry of these fundamental folds can be readily mimicked in peptidomimetics.     

Selective recognition of parallel and anti-parallel thrombin-binding aptamer G-quadruplexes by different fluorescent dyes

G-quadruplexes (G4) have been found increasing potential in applications, such as molecular therapeutics, diagnostics and sensing. Both Thioflavin T (ThT) and N-Methyl mesoporphyrin IX (NMM) become fluorescent in the presence of most G4, but thrombin-binding aptamer (TBA) has been reported as the only exception of the known G4-forming oligonucleotides when ThT is used as a high-throughput assay to identify G4 formation. Here, we investigate the interactions between ThT/NMM and TBA through fluorescence spectroscopy, circular dichroism and molecular docking simulation experiments in the absence or presence of cations. The results display that a large ThT fluorescence enhancement can be observed only when ThT bind to the parallel TBA quadruplex, which is induced to form by ThT in the absence of cations. On the other hand, great promotion in NMM fluorescence can be obtained only in the presence of anti-parallel TBA quadruplex, which is induced to fold by K⁺ or thrombin. The highly selective recognition of TBA quadruplex with different topologies by the two probes may be useful to investigate the interactions between conformation-specific G4 and the associated proteins, and could also be applied in label-free fluorescent sensing of other biomolecules.     

Centromere elasticity

In addition to the role in the spindle apparatus and associated motors, the chromosome themselves play an important role in facilitating chromosome segregation. Sister chromatids are joined at the centromere through a protein complex called cohesin. Chromatids separation requires the degradation by separase of specific proteins acting as a glue to form the cohesin complex. This evolutionally complex is required for the establishment and maintenance of sister chromatids in a ring like structure. It is therefore a key question whether cohesin is indeed a main component of active centromere. Cohesin is insufficient to resist the splitting force exerted by microtubules until anaphase and must be renforced by cohesion provided by flanking DNA. The ring model suggests that cohesine might possess a considerable mobility when associated with chromatin. Observations demonstrate that the interior region of the centromere behaves as an elastic element. Chromosomes display remarkable elasticity, returning to their initial shape after being extended by up to 10 times. For larger deformations the thick filament is converted in thin filament which can be stretched six times before breaking. This article suggests an additional and novel role for the protein titin on chromosome structure and dynamic. Titine was identified as a chromosomal component and it was hypothesised that titin may provide elasticity to chromosome and resistance to chromosome breakages during mitosis. The elastic properties of purified titin correspond well to the elastic properties of chromosome in living cells. The deformability and bending rigidity are consistent with a model developed for titin elasticity. The association of the presence of cohesine ring and the activity of titin could be necessary for segregation.     

Bending membranes

It is widely assumed that peripheral membrane proteins induce intracellular membrane curvature by the asymmetric insertion of a protein segment into the lipid bilayer, or by imposing shape by adhesion of a curved protein domain to the membrane surface. Two papers now provide convincing evidence challenging these views. The first shows that specific assembly of a clathrin protein scaffold, coupled to the membrane, seems to be the most prevalent mechanism for bending a lipid bilayer in a cell. The second reports that membrane crowding, driven by protein-protein interactions, can also drive membrane bending, even in the absence of any protein insertion into the bilayer.     

Folding of beta-sheets in membranes: specificity and promiscuity in peptide model systems

The interactions that drive the folding of beta-barrel membrane proteins have not been well studied because there have been few available model systems for membrane beta-sheets. In this work, we expand on a recently described model system to explore the contributions of interstrand hydrogen bonds, side-chain/side-chain interactions and side-chain/membrane interactions to beta-sheet formation in membranes. These experiments are based on the observation that the hydrophobic hexapeptide acetyl-Trp-Leu-Leu-Leu-Leu-Leu-OH (AcWLLLLL) folds, cooperatively and reversibly, into oligomeric, antiparallel beta-sheets in phosphatidylcholine membranes. To systematically characterize the important interactions that drive beta-sheet formation in membranes, we have used circular dichroism spectroscopy to determine the membrane secondary structure of each member of a complete host-guest family of related peptides of the form AcWLL-X-LL, where X is one of the natural amino acids. Peptides with hydrophobic X-residues of any size or character (X=Ala, Val, Ile, Leu, Cys, Met, Phe and Trp) form similar beta-sheets in membranes, while peptides with any polar X-residue or Gly or Pro at the X-position are random-coils, even when bound to membranes at high concentrations. The observed membrane sheet preferences correlate poorly with intrinsic sheet propensity scales measured in soluble proteins, but they correlate well with several membrane hydrophobicity scales. These results support the idea that the predominant interactions of the side-chains in membrane-bound beta-sheets are with the membrane lipids, and that backbone hydrogen bonding is the major driving force for the stabilization of beta-sheets in membranes.     

Peptidomimetics and the template approach to nucleation of beta-sheets and alpha-helices in peptides.

Introducing structural constraints into engineered analogs of natural proteins is important for defining essential characteristics, such as specificity or stability, of the protein. This may be achieved by the use of peptidomimetics--elements which mimic the structure of natural peptide components, or conformational templates which induce specific structure formation in contiguous peptide sequence.     

Role of interchain interactions in the stabilization of the right-handed twist of beta-sheets

Conformational energy computations have been carried out for parallel and antiparallel beta-sheets composed of poly-L-Val and poly-L-Ile peptide chains, each consisting of four and of six residues, respectively, with CH3CO- and-NHCH3 end groups. The beta-sheets considered contained three and five equivalent chains, respectively. All computed minimum-energy beta-sheets were found to have a large right-handed twist of a magnitude that corresponds to the mean twist of beta-sheets observed in globular proteins. The twist has the same sign but is much larger than in beta-sheets of poly-L-Ala, because of intra- and interchain interactions between the bulky beta-branched side-chains. While the right-handed twist is a result of intrachain interactions between side-chains in the case of poly-L-Val, these interactions would favor a left-handed twist in poly-L-Ile, and the right-handed twist in the latter is a result of interchain interactions. Parallel beta-sheets are more stable than antiparallel sheets for both poly-L-Val and poly-L-Ile, in contrast to poly-L-Ala. This result agrees with observations on the preferred orientation of the chains in oligopeptides that form beta-structures. It also explains the observed high relative frequencies of occurrence of Val and Ile residues in parallel beta-sheets, as compared with antiparallel sheets, in globular proteins.     

Double-stranded oligonucleotides containing 5-aminomethyl-2'-deoxyuridine form thermostable anti-parallel triplexes with single-stranded DNA or RNA

This Letter describes the synthesis and properties of double-stranded antisense oligonucleotides connected with a pentaerythritol linker. We found that double-stranded antisense oligonucleotides with aminomethyl residues have high affinity for single-stranded DNA or RNA in buffer solutions with and without MgCl(2). Thus, these oligonucleotides would be useful as antisense oligonucleotides for targeting single-stranded RNA through triplex formation.