Self-assembling peptide scaffolds promote enamel remineralization.

Rationally designed beta-sheet-forming peptides that spontaneously form three-dimensional fibrillar scaffolds in response to specific environmental triggers may potentially be used in skeletal tissue engineering, including the treatment/prevention of dental caries, via bioactive surface groups. We hypothesized that infiltration of caries lesions with monomeric low-viscosity peptide solutions would be followed by in situ polymerization triggered by conditions of pH and ionic strength, providing a biomimetic scaffold capable of hydroxyapatite nucleation, promoting repair. Our aim was to determine the effect of an anionic peptide applied to caries-like lesions in human dental enamel under simulated intra-oral conditions of pH cycling. Peptide treatment significantly increased net mineral gain by the lesions, due to both increased remineralization and inhibition of demineralization over a five-day period. The assembled peptide was also capable of inducing hydroxyapatite nucleation de novo. The results suggest that self-assembling peptides may be useful in the modulation of mineral behavior during in situ dental tissue engineering.

Electrochemical screening of self-assembling beta-sheet peptides using supported phospholipid monolayers.

In the context of the medical applications of beta-sheet self-assembling peptides, it is important to be able to predict their activity at the biological membrane level. A study of the interaction of four systematically varied 11-residue (P11-1, P11-2, P11-6 and P11-7) and one 13-residue (P13-1) designed beta-sheet self-assembling peptides with DOPC monolayers on a mercury electrode is reported in this paper. Experiments were carried out in 0.1 mol dm(-3) KCl electrolyte with added phosphate buffer (0.001 mol dm(-3)) at pH approximately 7.6. The capacity-potential curves of the coated electrode in the presence and absence of the different peptides were measured using out-of-phase ac voltammetry. The frequency dependence of the complex impedance of the coated electrode surfaces in the presence and absence of the peptides was estimated between 65,000 and 0.1 Hz at -0.4V versus Ag/AgCl 3.5 mol(-3) dm(-3) KCl. The monolayer permeabilising properties of the peptides were studied by following the reduction of Tl(I) to Tl(Hg) at the coated electrode. Of the five peptides studied, P11-2, P11-7 and P13-1 interact most strongly with the DOPC layer. P11-1 which has a polar primary structure shows no obvious interaction with the phospholipid but surprisingly, it permeabilises the phospholipid layer to Tl(+).

The internal dynamic modes of charged self-assembled peptide fibrils.

Photon correlation spectroscopy is used to study the internal dynamics of self-assembled charged peptide fibrils. Short neutral and charged polymeric aggregates have diffusive modes due to whole macromolecular motion. For long semiflexible fibrils the logarithm of the intermediate scattering function follows a q(2)t(3/4) scaling at long times consistent with a Kratky-Porod free energy and preaveraged Oseen hydrodynamics. Persistence lengths on the order of micrometers are calculated for the peptide fibrils consistent with estimates from the liquid-crystalline phase behavior. Fibril diameters (5-35 nm) calculated from the initial decay of the correlation functions are in agreement with transmission electron microscopy measurements.

Nature of the liquid crystalline phase transitions in the cesium pentadecafluorooctanoate-water system: the nematic-to-smectic-A transition.

Measurements of the diamagnetic susceptibility chi(a), the rotational viscosity coefficient gamma(1), and the micellar orientational order parameter S have been made as a function of temperature across the entire nematic phases of cesium pentadecafluorooctanoate (CsPFO)-D2O samples over the concentration range weight fraction CsPFO w from 0.350 to 0.500. This has been chosen so as to embrace the pseudo-nematic phase-lamellar tricritical point that has been purported to occur at w=0.43 (volume fraction phi=0.26). The values of gamma(1) vary from about 0.1 kg m(-1) s(-1) to 1000 kg m(-1) s(-1) as the temperature decreases across the nematic phase, covering a much larger range than those reported for thermotropic liquid crystals. Measurements of gamma(1) have been made much closer to the critical temperature T(LN) than have previously been reported [(T-T(LN))/T(LN)=2 x 10(-5) for the w=0.350 sample]. The rotational viscosity critical exponents x calculated from the divergence of gamma(1) on approaching the nematic-phase-to-lamellar transition are found to be constant at 2/3 independent of the concentration. These values differ from those of 1/3 and 1/2, respectively, predicted by the He-like three-dimensional XY and mean-field models, in combination with mean-field dynamical relaxation. However, the former would be supported if complete Fisher renormalization (at alpha=1/2) was appropriate over an extended concentration range. Alternatively, this could be an indication of the importance of coupling between the micellar rotational dynamics and critical fluctuations since the latter are seen to be predominant across the entire temperature range of the nematic phase as revealed in the temperature dependence of gamma(1).

Structure and dynamics of self-assembling beta-sheet peptide tapes by dynamic light scattering.

Oligomeric peptides can be designed which undergo one-dimensional self-assembly in solution to form beta-sheet tapes a single molecule in thickness and micrometers in length.(1) In this paper, we present the first systematic investigation of the size, shape, dynamics, and interactions of beta-sheet tapes formed by a self-assembling 24-residue peptide, K24, in 2-chloroethanol, over a wide range of peptide concentrations c (c: 10(-7)-1 mM), using photon correlation spectroscopy. The tapes behave like semiflexible chains with persistence lengths of several hundred nanometers and much longer contour lengths, even at c approximately 0.1 nM. The polarized q-dependent light-scattering intensity I fits a model of a prolate object with major and minor axes alpha approximately 630 nm and beta approximately 40 nm. This is an unexpected result in view of the previous theoretical predictions that tapelike polymers could form oblate coinlike structures in solution.(2) This experimentally observed behavior is attributed to the pronounced twist and bend of the beta-tapes, which do not allow them to form the coinlike structures, but instead they favor the formation of elongated polymers. At c approximately 10(-2) mM, the tapes are seen to start overlapping and forming networks with unusually large mesh sizes (e.g., ca. 400 nm at 15 mciroM), much larger than those of conventional polymers. With increasing peptide concentration the mesh size decreases and the network becomes a physical gel at c approximately 0.4 mM. These semidilute solutions are characterized by one main relaxation mode associated with the cooperative diffusion of the entangled tape network, and a weaker slower mode, associated with gel cluster formation. The concentration dependence of xi (xi(c) approximately c(-0.34)) is much weaker compared to the expected scaling for Gaussian or swollen chains (xi(c) approximately c(-1) for Gaussian chains, or xi(c) approximately c(-3/4) for swollen ones), but is not inconsistent with the expected scaling for rigid rods. On the basis of the concentration dependencies of the light-scattering intensity I and of the cooperative diffusion coefficient D, the cooperative friction coefficient f(c) is found to display a stronger concentration dependence (f(c) approximately c(1.34)) than in the case of semidilute flexible and semiflexible polymer solutions (f(c) approximately c(0.5)).(3) Thus, we may conclude that the network of entangled tapes approximates in its behavior that of semirigid polymers.

Hierarchical self-assembly of chiral rod-like molecules as a model for peptide beta -sheet tapes, ribbons, fibrils, and fibers.

A generic statistical mechanical model is presented for the self-assembly of chiral rod-like units, such as beta-sheet-forming peptides, into helical tapes, which with increasing concentration associate into twisted ribbons (double tapes), fibrils (twisted stacks of ribbons), and fibers (entwined fibrils). The finite fibril width and helicity is shown to stem from a competition between the free energy gain from attraction between ribbons and the penalty because of elastic distortion of the intrinsically twisted ribbons on incorporation into a growing fibril. Fibers are stabilized similarly. The behavior of two rationally designed 11-aa residue peptides, P(11)-I and P(11)-II, is illustrative of the proposed scheme. P(11)-I and P(11)-II are designed to adopt the beta-strand conformation and to self-assemble in one dimension to form antiparallel beta-sheet tapes, ribbons, fibrils, and fibers in well-defined solution conditions. The energetic parameters governing self-assembly have been estimated from the experimental data using the model. The 8-nm-wide fibrils consist of eight tapes, are extremely robust (scission energy approximately 200 k(B)T), and sufficiently rigid (persistence length l(fibril) approximately 20-70 microm) to form nematic solutions at peptide concentration c approximately 0.9 mM (volume fraction approximately 0.0009 vol/vol), which convert to self-supporting nematic gels at c > 4 mM. More generally, these observations provide a new insight into the generic self-assembling properties of beta-sheet-forming peptides and shed new light on the factors governing the structures and stability of pathological amyloid fibrils in vivo. The model also provides a prescription of routes to novel macromolecules based on a variety of self-assembling chiral units, and protocols for extraction of the associated energy changes.

Nature of the liquid crystalline phase transitions in the cesium pentadecafluorooctanoate (CsPFO)-water system: the nematic-to-isotropic transition.

Deuterium NMR spectroscopy of 2H2O has been used to monitor the magnetic-field-induced order on approaching a transition to a nematic phase in isotropic solutions of disklike micelles of cesium pentadecafluorooctanoate. Highly accurate data on the phase boundaries and spinodals have been obtained for solutions with volume fraction concentration straight phi between 0.078 and 0.201. The quantity TIN-T*, where T* is the spinodal limit of the isotropic phase and TIN is the temperature at which the nematic phase first appears on cooling, decreases linearly with decreasing concentration, extrapolating to zero only at zero concentration. Thus, there is no evidence to support the presence of a Landau point along the transition line as has previously been conjectured. The values for (TIN-T*)/TIN are in the range 10(-5)-10(-4), up to two orders of magnitude smaller than corresponding values reported for calamitic thermotropic nematics. The transition gap (phiNI-phiIN)/phiIN approximately 0.33% for phi<0.20 is also very small, although finite as required for a first-order phase transition. These data, when combined with previously measured properties, present an intriguing picture of the isotropic-to-nematic phase transition in a paradigmatic system of self-assembled diskotic particles. However, it is not completely clear, within the context of current theoretical understanding, whether the behavior of this system is explicable by hard-particle models, or if the self-assembly plays a crucial role in weakening the phase transition.

Conformation and ion-channeling activity of a 27-residue peptide modeled on the single-transmembrane segment of the IsK (minK) protein.

IsK (minK) protein, in concert with another channel protein KVLQT1, mediates a distinct, slowly activating, voltage-gated potassium current across certain mammalian cell membranes. Site-directed mutational studies have led to the proposal that the single transmembrane segment of IsK participates in the pore of the potassium channel [Takumi, T. (1993) News Physiol. Sci. 8, 175-178]. We present functional and structural studies of a short peptide (K27) with primary structure NH2-1KLEALYILMVLGFFGFFTLGIMLSYI27R-COOH, corresponding to the transmembrane segment of IsK (residues 42-68). When K27 was incorporated, at low concentrations, into phosphatidylethanolamine, black-lipid membranes, single-channel activity was observed, with no strong ion selectivity. IR measurements reveal the peptide has a predominantly helical conformation in the membrane. The atomic resolution structure of the helix has been established by high-resolution 1H NMR spectroscopy studies. These studies were carried out in a solvent comprising 86% v/v 1,1,1,3,3,3-hexafluoro-isopropanol-14% v/v water, in which the IR spectrum of the peptide was found to be very similar to that observed in the bilayer. The NMR studies have established that residues 1-3 are disordered, while residues 4-27 have an alpha-helical conformation, the helix being looser near the termini and more stable in the central region of the molecule. The length (2. 6 nm) of the hydrophobic segment of the helix, residues 7-23, matches the span of the hydrocarbon chains (2.3 +/- 0.25 nm) of fully hydrated bilayers of phosphatidylcholine lipid mixture from egg yolk. The side chains on the helix surface are predominantly hydrophobic, consistent with a transmembrane location of the helix. The ion-channeling activity is believed to stem from long-lived aggregates of these helices. The aggregation is mediated by the pi-pi stacking of phenylalanine aromatic rings of adjacent helices and favorable interactions of the opposing aliphatic-like side chains, such as leucine and methionine, with the lipid chains of the bilayer. This mechanism is in keeping with site-directed mutational studies which suggest that the transmembrane segment of IsK is an integral part of the pore of the potassium channel and has a similar disposition to that in the peptide model system.

Laser-Raman and FT-IR spectroscopic studies of peptide-analogues of silkmoth chorion protein segments.

Silkmoth chorion, the proteinaceous major component of the eggshell, with extraordinary mechanical and physiological properties, consists of a complex set of proteins, which have a tripartite structure: a central, evolutionarily conserved, domain and two more variable 'arms'. Peptide-analogues of silkmoth chorion protein central domain segments have been synthesized. Laser-Raman and infrared spectroscopic studies suggest the preponderance of antiparallel beta-pleated sheet structure for these peptides, both in solution and in the solid state.

Equilibrium and non-equilibrium conformations of peptides in lipid bilayers.

A synthetic, hydrophobic, 27-amino-acid-residue peptide 'K27', modelled on the trans-membrane domain of the slow voltage-gated potassium channel, IsK, has been incorporated into a lipid bilayer and its conformational properties studied using FT-IR spectroscopy. The conformation following reconstitution is found to be dependent on the nature of the solvent employed. When the reconstitution is conducted by solvent evaporation from a methanol solution, aggregates comprised of beta-strands are stabilised and their concentration is essentially invariant with time. By contrast, when trifluoroethanol is used, the initial conformation of the peptide is alpha-helical. This then relaxes to an equilibrium state between alpha-helices and beta-strands. The alpha-helix-to beta-strand conversion rate is relatively slow, and this allows the kinetics to be studied by FT-IR spectroscopy. The reverse process is much slower but again can be demonstrated by FT-IR. Thus, it appears that a true equilibrium structure can only be achieved by starting with peptide in the alpha-helical conformation. We believe this result should be of general validity for hydrophobic peptide reconstitution. The implications for conformational changes in membrane proteins are discussed.

Responsive gels formed by the spontaneous self-assembly of peptides into polymeric beta-sheet tapes.

Molecular self-assembly is becoming an increasingly popular route to new supramolecular structures and molecular materials. The inspiration for such structures is commonly derived from self-assembling systems in biology. Here we show that a biological motif, the peptide beta-sheet, can be exploited in designed oligopeptides that self-assemble into polymeric tapes and with potentially useful mechanical properties. We describe the construction of oligopeptides, rationally designed or based on segments of native proteins, that aggregate in suitable solvents into long, semi-flexible beta-sheet tapes. These become entangled even at low volume fractions to form gels whose viscoelastic properties can be controlled by chemical (pH) or physical (shear) influences. We suggest that it should be possible to engineer a wide range of properties in these gels by appropriate choice of the peptide primary structure.

Peptides modeled on the transmembrane region of the slow voltage-gated IsK potassium channel: structural characterization of peptide assemblies in the beta-strand conformation.

A 27-residue peptide, having a sequence corresponding to the transmembrane domain of the IsK protein with slow voltage-gated potassium channel activity, has been incorporated into synthetic saturated-chain phospholipid membranes. The peptide-lipid complexes have been characterized by attenuated-total-reflection Fourier-transform-infrared spectroscopy (ATR-FTIR), spin-label electron spin resonance (ESR) spectroscopy, 31P and 2H nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry, and low-angle X-ray diffraction. From FTIR spectroscopy, it is found that, when reconstituted into membranes by dialysis from 2-chloroethanol, the peptide has a predominantly beta-strand secondary structure in which the peptide backbone is oriented at an angle of approximately 56 degrees relative to the membrane normal in dry films of phosphatidylcholines. Hydration of the dry film in the gel phase does not appear to affect the orientation of the peptide backbone, and a relatively small change in orientation occurs when the bilayer undergoes the transition to the fluid phase. The ESR and NMR spectra from spin-labeled and 2H-labeled phospholipids, respectively, indicate that the incorporated peptide restricts the rotational motion of the lipids, without appreciably affecting the chain order, in a way similar to that found for integral membrane proteins. The characteristic two-component ESR spectra from spin-labeled lipids further indicate a selectivity in the interaction of anionic phospholipids with the peptide. The motional restriction of the chains of the spin-labeled phosphatidylcholine and the reduction in the enthalpy of the lipid chain-melting transition indicate that, on average, approximately two to three phospholipid molecules interact directly with each peptide monomer, which is consistent with a limited degree of aggregation of the beta-sheet structures. Both 31P NMR spectroscopy and X-ray diffraction indicate that the lipid-peptide complexes have a lamellar structure up to the highest peptide concentration studied (Rp = 0.2). The surface area occupied by lipid molecules (ca. 30 A2 per chain) in the peptide complexes, deduced from the lamellar repeat spacings at defined water content, is very similar to that in pure fluid lipid bilayers, consistent with the 2H NMR results. The additional membrane surface area contributed by the peptide is approximately 112 A2 per monomer. This large value for the peptide area in the fluid bilayer is consistent with the ATR studies of dry peptide/lipid films which suggest that the long axis of the beta-strand is strongly tilted with respect to the bilayer normal (56 degrees in the dry film).

The conformational behaviour of phosphatidylinositol in model membranes: 2H-NMR studies.

Dimyristoylphosphatidylinositol (DMPI) has been synthesized with the appropriate natural stereochemistry and labelled with deuterium at specific sites in the D-myo-inositol headgroup. 2H-NMR spectroscopy of DMPI in its lamellar phase at a molar ratio of water-to-lipid RW/L of 129 and at 70 degrees C reveals quadrupolar splittings delta v of 3.83 and 2.17 kHz, respectively, for the five axially oriented C-D bonds and the single equatorially oriented C-D bond of the D-myo-inositol headgroup. Between RW/L ratios of 129 and 210 and between 30 degrees C and 80 degrees C the value of the ratio of these splittings delta nu ax/delta nu eq varies significantly (between 1.17 and 4.38). If it is assumed that, at a particular temperature, there is a single preferred orientation of the inositol headgroup, and that motion of the DPMI molecule establishes axial symmetry with respect to the bilayer normal then the ratio of these quadrupolar splittings can be used to impose constraints on that orientation. For example, the data are inconsistent with a situation in which the inositol ring lies parallel to the membrane surface and are difficult to reconcile with an arrangement where the inositol ring lies perpendicular to the surface. Computational modelling identifies four possible 'tilted' orientations, all of which are consistent with the data, and two of these allow good intramolecular hydrogen bonds to be formed. In one there is hydrogen bonding between the inositol C2-OH and the phosphate pro-R oxygen. This is close to the conformation previously identified as being dominant in DMSO solution (Bushby, R.J., Byard, S.J., Hansbro, P.M. and Reid, D.G. (1990) Biochim. Biophys. Acta 1044, 231-236).

On the use of deuterium nuclear magnetic resonance as a probe of chain packing in lipid bilayers.

The packing of hydrocarbon chains in the bilayers of lamellar (L alpha) phases of soap/water and phospholipid/water mixtures has been studied by deuterium NMR spectroscopy and X-ray diffraction. A universal correlation is shown to exist between the average C-D bond order parameter SCD of hydrocarbon chains and the average area per chain ach, irrespective of the chemical structure of the surfactant (hydrophilic group, number of chains per molecule, and chain length), composition, and temperature. The practical utility of the correlation is illustrated by its application to the characterization of the distribution of various hydrophobic and amphiphilic solutes in bilayers. The distribution of hydrocarbons within a bilayer is shown to depend upon their molecular structure in a manner which highlights the nature of the molecular interactions involved. For example, benzene is shown to be fairly uniformly distributed across the bilayer with an increasing tendency to distribute into the center at high concentrations. In contrast, the more complex hydrocarbon tetradecane preferentially distributes into the center of the bilayer at low concentrations, while at higher concentrations it intercalates between the surfactant chains. Alcohols such as benzyl alcohol, octanol, and decanol all interact similarly with the bilayer in so far as they are pinned to the polar/apolar interface, presumably by involvement of the hydroxyl group in a hydrogen bond. But the response of the surfactant chains to the void volume created in the center of the bilayer is dependent upon the distance of penetration of the alcohol into the bilayer. For benzyl alcohol, the shortest molecule, this void volume is taken up by the disordering of the chains, while for decanol, the longest molecule, it is absorbed by interdigitation of the chains of apposing monolayers. For octanol, the chain interdigitation mechanism is dominant at low concentrations, but there is a transition to chain disordering at high concentrations. Finally, it is shown that the correlation provides a useful test for statistical mechanical models of chain ordering in lipid bilayers.

Properties of the cupric sites in bovine superoxide dismutase studied by nuclear-magnetic-relaxation measurements.

Proton nuclear-relaxation rates have been measured as a function of frequency, temperature, pH and cyanide concentration in aqueous solutions of superoxide dismutase from bovine erythrocytes. The results show that, whereas for pH less than or equal to 9 only one water molecule is bound to each Cu2+ ion, at higher pH a second co-ordination site for OH- becomes available; it is proposed that this involves cleavage of the bond between Cu2+ and the histidine residue that bridges to Zn2+.