Effects of Na2SO4 on hydrophobic and electrostatic interactions between amphipathic alpha-helices.

The effects of 2 molal Na2SO4 at neutral pH on hydrophobic and electrostatic interactions between amphipathic alpha-helices were investigated by circular dichroism spectroscopy. The amphipathic peptides that were studied included LEK (acetyl-LEELKKKLEELKKKLEEL-NH2) and LEE (acetyl-LEELEEELEELEEELEEL-NH2). In phosphate buffer at neutral pH, only LEK adopted a predominantly alpha-helical conformation, attributable to glu-lys+ interactions where a major contribution is evidently a hydrogen bond (Biochemistry 32: 9668-9676). Despite the presence of lys+ in the e and g' positions of the abcdefg heptad repeat, LEK exhibited mean-residue ellipticities at 222 nm ([theta]222) which were dependent on peptide concentration, indicating the presence of a coiled coil. In the presence of 2 molal Na2SO4 at 25-75 degrees C, the helical content of LEK increased, with the greatest increase observed at 75 degrees C. The value of the ellipticity ratio R ([theta]222/[theta]208) of LEK in 2 molal Na2SO4 also increased, indicating a stronger interhelical association. At 50 degrees C and 75 degrees C, LEK remained predominantly alpha-helical. In phosphate buffer at neutral pH, LEE was mainly random coil. In the presence of 2 molal Na2SO4, however, the peptide formed alpha-helices that associated to form a coiled coil. At 50 degrees C and 75 degrees C, LEE became predominantly random coil but the remaining alpha-helices were still associating. These results are consistent with the strengthening of interhelical hydrophobic interactions and the absence of screening of helix-stabilizing and helix-destabilizing electrostatic interactions in amphipathic alpha-helices by Na2SO4.

A mixture of alpha-helical and 3(10)-helical conformations for involucrin in the human epidermal corneocyte envelope provides a scaffold for the attachment of both lipids and proteins.

Involucrin plays an important role in the lipid and protein compound envelopes of mammalian epidermal corneocytes. In the present study, model peptides containing the consensus repeating units PEQQEGQLEL and LEQQEGQLEH, found in the central region of human involucrin, were studied by circular dichroism spectroscopy, molecular modeling, and energy minimization. These peptides have intrinsic alpha-helix-forming properties as indicated by their circular dichroic spectra obtained in the presence of 2,2,2-trifluoroethanol. Peptide (LEQQEGQLEH)(3) had an alpha-helix content of 100% in 100% 2, 2,2-trifluoroethanol at 0 degrees C. The energy-minimized alpha-helix showed that only 50% of the glutamate side chains may be available for the attachment of lipids. However, when a 3(10)-helix was assumed for the GQL or GQLE residues in LEQQEGQLEH, all of the glutamate side chains were arrayed on one face of the helix, and all of the glutamine side chains were arrayed on the opposite face. A similar result was obtained when the nonhelical part of PEQQEGQLEL was assumed to contain a beta-turn III, which is equivalent to a short portion of 3(10)-helix. The results of this study suggest that when the central segment of human involucrin is predominantly alpha-helical, accompanied by short 3(10)-helical segments, the protein can function as a scaffold for the attachment of both lipids and proteins.

Molecular modelling indicates that the pathological conformations of prion proteins might be beta-helical.

Creutzfeldt-Jakob disease, kuru, scrapie and bovine spongiform encephalopathy are diseases of the mammalian central nervous system that involve the conversion of a cellular protein into an insoluble extracellular isoform. Spectroscopic studies have shown that the precursor protein contains mainly alpha-helical and random-coil conformations, whereas the prion isoform is largely in the beta conformation. The pathogenic prion is resistant to denaturation and protease digestion and can promote the conversion of the precursor protein to the pathogenic form. These properties have yet to be explained in terms of the structural conformations of the proteins. In the present study, molecular modelling showed that prion proteins could adopt the beta-helical conformation, which has been established for a number of fibrous proteins and has been suggested previously as the basis of amyloid fibrils. The beta-helical conformation provides explanations for the biophysical and biochemical stability of prions, their ability to form templates for the transmission of pathological conformation, and the existence of phenotypical strains of the prion diseases.

Fibril formation by amyloid-beta proteins may involve beta-helical protofibrils.

We have proposed that amyloid fibrils contain subunits (protofibrils) that are formed from beta-strands wound into continuous 2-3 nm-diameter beta-helices. Subsequent lateral aggregation of the beta-helices to form the widely observed 5-12 nm-diameter fibrils could be promoted by hydrophobic residues on the exterior of the postulated beta-helix. A number of short peptide fragments of the amyloid-beta (A beta) proteins, such as A beta34-42 [LMVGGVVIA], the nine-residue, carboxyl-terminal portion of A beta1-42, can also form amyloid fibrils. In the present study, it was found that a beta-helix formed from A beta34-42 accounts for features suggested by published rotational resonance solid-state NMR data, including an anomalous conformation about the Gly-37-Gly-38 region and exaggerated pleating. An analogue of A beta34-42 was synthesized in which the hydrophobic groups on the exterior of the postulated beta-helix were replaced with glutamates, giving LEVGGVEIE. The analogue was completely soluble at pH 7, but at pH 2.5 it produced 2-2.5 nm-diameter fibrils which did not associate into larger-diameter bundles. The results of this study support the proposal that amyloid fibrils are formed from beta-helical subunits.

Stabilization of amphipathic alpha-helical and beta-helical conformations in synthetic peptides in the presence and absence of ionic interactions.

The role of ionic interactions in stabilizing amphipathic alpha-helices was studied in the synthetic peptide Ac-NLEELKKKLEELKG-NH2 (NLEKG14), potentially stabilized by attraction between complementary ions in successive turns of the helix, and in the peptide Ac-NLEELEEELEELEG-NH2 (NLEG14), in which no side-chain ionic attractions are possible. At a pH below the pKa of glutamate, NLEG14 had a higher helix content than NLEKG14. At pH 3 to pH 10, the helicity of NLEKG14 did not change, whereas NLEG14 was converted to random coil at pH 7. The role of ionic interactions in stabilizing the conformation of beta-structures was studied in the synthetic peptides Ac-KLKLKLELELELG-NH2 (KLEG13) and Ac-ELELELELELELG-NH2 (ELG13). At a pH below the pKa of glutamate, ELG13 had a higher beta-content than KLEG13, as judged by their dichroic spectra, but at higher pH, ELG13 was converted to random coil, whereas KLEG13 retained a predominantly beta-conformation. At pH 7, high NaCl concentration produced a significant increase in the alpha-helix content of NLEKG14, converted NLEG14 from random coil to alpha-helix and converted ELG13 from random coil to beta-conformation. Overall, the results demonstrate that ionic attraction between side-chains plays a lesser role than hydrogen bonding and hydrophobic effects in stabilizing the alpha- and beta-conformations exhibited by these amphipathic peptides.

Beta-helical fibrils from a model peptide.

A synthetic peptide, KLEG13 (Ac-KLKLKLELELELG-NH2), composed of alternating bulky hydrophilic and hydrophobic amino acid residues formed clear, viscous dispersions of fibrils in saline solutions. The fibrils had a uniform diameter of 2 nm as measured on electron micrographs of negatively stained preparations. 13C solid-state nuclear magnetic resonance spectroscopy of the fibrils indicated the presence of a beta-conformation. Circular dichroic spectra of the dispersion of fibrils were essentially identical to the calculated spectrum of a 100% beta-helix. Space-filling CPK models of a proposed beta-helical conformation of the peptide, in which the leucine side chains form a hydrophobic core and the hydrophilic lysine and glutamate side chains extend outwards from the helix, had a diameter consistent with the observed 2-nm diameter of the fibrils. This study may have implications regarding the structure of amyloid fibrils.

Circular dichroism of model peptides emulating the amphipathic alpha-helical regions of intermediate filaments.

The a and d positions of the heptad repeats (abcdefg) found in the alpha-helical sections of intermediate-filament proteins are hydrophobic, and the remaining locations are almost exclusively hydrophilic and often charged. Two synthetic peptides that maximize these features were designed, synthesized, and investigated by circular dichroism for alpha-helix formation in water and in 50% trifluoroethanol (TFE). A 14-residue peptide, AcNLEELKKKLEELKGNH2 (NLEKG14), had mean residue ellipticities at 222 nm ([theta]222) of -18400 +/- 1000 and -37,200 +/- 1900 deg cm2 dmol(-1), in water at 2 degrees C and in 50% TFE at 2 degrees C, respectively. A longer version of NLEKG14, AcNLEELKKKLEELKQQLEELKKKLEELKQQNH2 (NLEKQ29), had [theta]222 of -43,000 +/- 2200 deg cm2 dmol(-1) in water and in 50% TFE at 2 degrees C. Using -43,000 deg cm2 dmol(-1) as [theta]222 for a 100% helix, NLEKG14 in 50% TFE at 25 degrees C was estimated to be 77% helix. This estimate was confirmed by two-dimensional 1H NMR studies of NLEKG14 in 50% TFE. Comparison with the sequences and conformations found in IF proteins indicates that the alpha-helical regions in the proteins may be exceptionally stable, but the high values for the ellipticity of alpha-helices now revealed allow for significant portions of the protein rod regions to be occupied by conformations other than alpha-helix.

Tryptophan dynamics and structural refinement in a lipid bilayer environment: solid state NMR of the gramicidin channel.

Tryptophans in the gramicidin channel are important for defining the conformation and the orientation with respect to the bilayer normal and for facilitating cation conductance. Here, high-resolution structure and dynamics of these rings are characterized by solid state NMR. Both oriented and unoriented lipid bilayer preparations are used. Fast frozen lipid bilayer preparations of unoriented samples have been used to obtain static characterizations of nuclear spin interaction tensors. The temperature dependence of these unoriented samples and the spectral features of fast frozen oriented samples were used to experimentally define the local motions of the individual indole rings. Local motions were shown to have amplitudes as high as +/- 29 degrees, and the motions were dominated by libration about the chi 2 axis. The high-resolution structure has been achieved by interpreting seven precise (+/- 0.3 degree) orientational constraints from 2H and 15N NMR for each indole ring in light of the motionally averaged interaction tensors. Each of the four indoles is restricted to a unique orientation of the ring with respect to the bilayer normal and one of two possible rotameric states. The side chain torsion angles for each residue are very similar, generating similar electric dipole moment orientations with respect to the channel.

Lipids are covalently attached to rigid corneocyte protein envelopes existing predominantly as beta-sheets: a solid-state nuclear magnetic resonance study.

C solid-state nuclear magnetic resonance at natural abundance was used to study isolated corneocyte envelopes from porcine stratum corneum. The presence of lipids covalently attached to the protein envelopes was detected by chemical shifts of methylene and methyl groups of the bound lipids. The corneocyte protein envelopes are rigid, as suggested by efficient 1H to 13C cross polarization and 13C spin-lattice relaxation studies. The chemical shift of the carbonyl carbons of the protein envelopes supports the prediction that the chemically bound lipid envelope is attached to proteins arranged predominantly in the beta-sheet conformation, allowing a dense palisade of ceramide molecules to form a water-impermeable external sheath.

Low-temperature solid-state 15N NMR characterization of polypeptide backbone librations.

The local molecular dynamics of gramicidin A in dimyristoylphosphatidylcholine bilayers were probed by a combination of low-temperature solid-state 15N NMR and rapid freezing of samples using liquid propane. It has previously been shown that this approach leads to sharp discontinuities in powder-pattern spectra of the gramicidin channel in lipid bilayers, and hence, an accurate determination of tensor element magnitudes is achieved. The static-tensor-element magnitudes determined here using hydrated bilayer samples at low temperatures are different from the values obtained from dry-powder samples at 308 K. However, for the one site studied, the orientation of the 15N chemical-shift tensor in the molecular frame is the same for both types of samples. Averaging of the chemical-shift tensor between 200 and 263 K is shown to be anisotropic and consistent with a motional axis parallel with the C alpha-C alpha axis for adjacent residues. The librational amplitudes determined here range from +/- 14 degrees to +/- 22 degrees at 263 K and are significantly larger than the nanosecond librations characterized by relaxation studies. The nanosecond motions are thought to represent correlated motions, while the librations described here are the sum of correlated (nanosecond) and uncorrelated (picosecond) motions. The picosecond librational amplitudes become considerably larger and more isotropic above 0 degree for the residues near the bilayer surface.

6-Hydroxy-4-sphingenine in human epidermal ceramides.

The solvent-extractable lipids of human epidermal stratum corneum consist predominantly of ceramides. In addition two non-extractable ceramides are chemically bound to the stratum corneum protein. One of the bound ceramides, constituting 50% of the bound lipids, was previously shown to consist of very long chan omega-hydroxyacids in amide linkage with sphingosine. The second bound caramide, which forms 25% of the bound lipids, was shown to contain the same hydroxyacids, but the sphingoid base was neither sphingosine nor phytosphingosine. In the present study, the undefined bound ceramide was shown by NMR and chemical procedures to be the omega-hydroxyacid derivative of a new base, 6-hydroxy-4-sphingenine. In addition, a ceramide previously known to constitute 25% of the extractable human stratum corneum ceramides has been found to contain the same novel sphingoid base, amide-linked to long-chain alpha-hydroxyacids. Finally, a new acylceramide has been isolated and identified that consists of very long chain omega-hydroxyacids in amide linkage with the novel sphingolipid, with fatty acids esterified wit the terminal hydroxyl group of the hydroxyacid.

Rapidly-frozen polypeptide samples for characterization of high definition dynamics by solid-state NMR spectroscopy.

A method is described for defining anisotropic local dynamics in polypeptides by solid-state NMR. To avoid conformational heterogeneity introduced by large hexagonal ice crystals in low temperature hydrated samples, a fast-freezing technique is used for sample preparation. For a demonstration of this approach, the backbone librational motions of the gramicidin A channel conformation are studied in hydrated DMPC bilayers. The static 15N chemical shift tensor is characterized at 123 K for the Ala3 site. The temperature dependence of this tensor yields a determination of the librational amplitude and anisotropy of the motionally sampled space. This amplitude represents the sum of nanosecond and picosecond time-frame motions, both of which have a significant amplitude.