Antiparallel Beta-sheet Structure Research Articles

The conformational analysis of peptides using fourier transform IR spectroscopy

Fourier transform infrared spectroscopy (FTIR) can be used for conformational analysis of peptides in a wide range of environments. Measurements can be performed in aqueous solution, organic solvents, detergent micelles as well as in phospholipid membranes. Information on the secondary structure of peptides can be derived from the analysis of the strong amide I band. Orientation of secondary structural elements within a lipid bilayer matrix can be determined by means of polarized attenuated total reflectance-FTIR spectroscopy. Hydrogen-deuterium exchange can be monitored by the analysis of the amide II band. This review gives some example of peptide systems studied by FTIR spectroscopy. Studies on alamethicin and alpha-aminoisobutyric acid containing peptides have shown that FTIR spectroscopy is a sensitive tool for identifying 3(10)-helical structures. Changes in the structure of the magainins upon interaction with charged lipids were detected using FTIR spectroscopy. Tachyplesin is an example of a beta-sheet containing membrane active peptide. Polarized ir spectroscopy reveals that the antiparallel beta-sheet structures of tachyplesin are oriented parallel to the membrane surface. Synthesis of peptides corresponding to functionally/structurally important regions of large proteins is becoming increasingly popular. FTIR spectroscopy has been used to analyze the structure of synthetic peptides corresponding to the ion-selective pore of the voltage-gated potassium channel. In biomembrane systems these peptides adopt a highly helical structure. Under conditions, where these peptides are aggregated the presence of some intermolecular beta-sheet structure can also be detected.

Subunit association and structural analysis of platelet basic protein and related proteins investigated by 1H NMR spectroscopy and circular dichroism.

Platelet basic protein (PBP) (94 residues) is naturally processed via N-terminal cleavage to yield connective tissue activating peptide-III (85 residues), beta-thromboglobulin (81 residues), and neutrophil activating peptide-2 (70 residues). Chemical cross-linking and gel filtration data indicate that each homolog can form dimers and tetramers. Subunit association equilibria for dimer (KD) and tetramer (KT) formation have been derived for each species from 1H NMR (600 MHz) spectral analysis of slowly exchanging (NMR time scale) monomer- dimer-tetramer aggregation state populations. In general, raising the pH from about pH 3.5 to pH 6 increases KD by two to three orders in magnitude and decreases KT by some 50-fold. Ionic strength effects also suggest that intersubunit electrostatic interactions are critical to subunit association. Subunit stabilization can be ranked proportional to N-terminal chain length: platelet basic protein > connective tissue activating peptide-III > beta-thromboglobulin > neutrophil activating peptide-2. Under more physiologic conditions, PBP family monomers are favored at normal cytokine protein concentrations and may form the biologically active state. CD and NMR data indicate conservation of alpha-helix and anti-parallel beta-sheet structure among PBP-related species and support the idea that the extended N terminus folds over and masks the neutrophil activation domain and is part of the intersubunit binding domain.

Reversible random coil-beta-sheet transition of the Alzheimer beta-amyloid fragment (25-35).

The beta-amyloid protein (39-43 amino acid residues) is the major constituent of the amyloid deposits found in brain of patients with Alzheimer's disease. Using circular dichroism spectroscopy, we have studied the secondary structure and the aggregation of fragment 25-35 of the beta-amyloid protein (beta AP(25-35)OH) under a variety of conditions. beta AP(25-35)OH in solution at pH 4.0 or 5.5 exhibits a concentration-dependent random coil<-->beta-sheet transition. The equilibrium is characterized spectroscopically by an isodichroic point and can be described quantitatively by a simple association model with association constants between 1.8 x 10(4) M-1 (non-cooperative model, nucleation parameter sigma = 1) and 2.9 x 10(4) M-1 (cooperative model, sigma = 0.2). The enthalpy of association is delta H approximately -3 kcal/mol as determined by titration calorimetry. The equilibrium is shifted completely toward beta-structured fibrils at pH 7.4 where the Met-35 carboxyl group is fully charged. In contrast, removal of the charged carboxy terminus by amidation locks the equilibrium in the random coil conformation. Model calculations suggest an antiparallel beta-sheet structure involving residues 28-35 which is stabilized at both ends of the beta-sheet by ion pairs formed between Lys-28 and Met-35. Removal of fibrils via millipore filtration leads to solutions with random coil monomers only. Seeding these solutions with a few fibrils establishes a new random coil<-->beta-sheet equilibrium.

Purification and characterization of beta-structural domains of beta-lactoglobulin liberated by limited proteolysis.

Incubation of beta-lactoglobulin with immobilized trypsin at 5-10 degrees C results in a time-dependent release of several fragments of the core domain in yields approaching 15%. Digests were fractionated by ion-exchange chromatography with a Mono Q HR 5/5 column and analyzed after disulfide reduction by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Three fragments with approximate molecular weights of 13.8, 9.6, and 6.7 kD were identified. The fraction from ion-exchange chromatography yielding the 6.7 kD fraction after disulfide reduction was further characterized because it was most homogeneous and gave the highest yield. The C-terminal cleavage site of the 6.7 kD core fragment appeared to be Lys100 or Lys101 as determined by C-terminal amino acid analysis. The exact masses, after reduction with dithiothreitol, are 6195 and 6926 as determined by laser desorption mass spectrometry, corresponding to residues 48-101 and 41-100. Prior to reduction, beta-lactoglobulin C-terminal residues 149-162 are connected to these core domain fragments as shown by C-terminal analysis and mass spectrometry. Structural studies indicate that these 7.9 and 8.6 kD core domain fragments released by immobilized trypsin retain much of their native structure. CD spectra indicate the presence of antiparallel beta-sheet structure similar to the native protein but the alpha-helix is lost. Spectra in the aromatic region indicate the existence of tertiary structure. Moreover, structural transitions in urea are completely reversible as measured by CD spectra, although the extrapolated delta GDH20 and the urea concentration at the transition midpoint are lower than for the native protein. The core domain fragments also display a pH-dependent binding to immobilized trans-retinal as does intact protein. A single endotherm is obtained for both core domain fragments and native protein upon differential scanning calorimetry, but again, the domain is less stable as indicated by a transition peak maxima of 56.9 degrees C as compared with 81.1 degrees C for native protein.

Desmoyokin, a 680 kDa keratinocyte plasma membrane-associated protein, is homologous to the protein encoded by human gene AHNAK

We have obtained a monoclonal antibody (33A-3D) that specifically recognize desmoyokin, a 680 kDa desmosomal plaque protein that is well characterized in bovine muzzle epidermis. A cDNA clone (DY6, 3693 bp) was isolated by immunoscreening a mouse keratinocyte expression library with 33A-3D, and it was confirmed that DY6 has a partial coding sequence for desmoyokin. DY6 consists of highly homologous repeats about 128 residues long. Furthermore, the 128-residue repeats exhibit a quasi seven-residue substructure, which we believe will adopt an antiparallel beta-sheet structure. Surprisingly, the amino acid sequence showed a significant homology with AHNAK, a newly identified human gene encoding a 700 kDa protein, which was suggested to be down-regulated in neuroblastoma. From its extensive homology, the similarity in both size and structure, and the identical patterns on Southern blot analysis of genomic DNAs, desmoyokin and AHNAK protein are thought to be identical. Although the desmoyokin/AHNAK protein is detected in a variety of cell types at both protein and mRNA levels, its distribution in keratinocytes (associated closely with cell membrane) is quite different from that in cells other than keratinocytes (distributed diffusely in the cytoplasm). These findings suggest that the desmoyokin/AHNAK protein is a ubiquitous molecule with a unique structure and appears to have different distributions (and probably different functions) among different cells.

Role of disulfide linkages in tachyplesin-lipid interactions.

In order to elucidate the role of the two disulfide linkages of tachyplesin I (T-SS), a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus, we synthesized the acyclic analog (T-Acm) with the four SH groups protected by acetamidomethyl groups and also investigated the interactions of these peptides with lipid bilayers. T-SS induced leakage of calcein from egg yolk L-alpha-phosphatidylglycerol large unilamellar vesicles (PG LUVs) at peptide concentrations 1 order of magnitude smaller than those at which leakage was induced by T-Acm, which coincides with the stronger antimicrobial activities of T-SS. The micellization of PG LUVs was also more efficient for the cyclic peptide. Fluorescence titration studies revealed that binding affinities of both peptides to the PG membranes were similar. Fourier transform infrared polarized attenuated total reflection spectroscopy and fluorescence quenching experiments demonstrated that T-SS and T-Acm both form amphiphilic antiparallel beta-sheet structures in the membranes. They are formed in such a way that the sheet planes lie parallel to the membrane surface with the sheet hydrophobic surfaces penetrating slightly into the hydrophobic region of the bilayers. Furthermore, the observation that the linear T-Acm, the weaker membrane permeabilizer, caused a far more serious membrane disruption suggests the possibility that the mechanisms of membrane permeabilization by the cyclic peptide are different from those by the linear peptide, the latter being the disruption of the lipid organization.

Crystal structure of tert.‐butyloxycarbonyl‐L‐prolyl‐D‐alanyl‐D‐alanyl‐N‐methylamide Dimeric β‐sheet formation

The crystal structure of the tripeptide t-Boc-L-Pro-D-Ala-D-Ala-NHCH3, monohydrate, (C17H30N4O5.H2O, molecular weight = 404.44) has been determined by single crystal X-ray diffraction. The crystals are monoclinic, space group P2(1), a = 9.2585(4), b = 9.3541(5), c = 12.4529(4)A, beta = 96.449(3) degrees, Z = 2. The peptide units are in the trans and the tBoc-Pro bond in the cis orientation. The first and third peptide units show significant deviations from planarity (delta omega = 5.2 degrees and delta omega = 3.7 degrees, respectively). The backbone torsion angles are: phi 1 = -60 degrees, psi 1 = 143.3 degrees, omega 1 = -174.8 degrees, phi 2 = 148.4 degrees, psi 2 = -143.1 degrees, omega 2 = -179.7 degrees, phi 3 = 151.4 degrees, psi 3 = -151.9 degrees, omega 3 = -176.3 degrees. The pyrrolidine ring of the proline residue adopts the C2-C gamma conformation. The molecular packing gives rise to an antiparallel beta-sheet structure formed of dimeric repeating units of the peptide. The surface of the dimeric beta-sheet is hydrophobic. Water molecules are found systematically at the edges of the sheets interacting with the urethane oxygen and terminal amino groups. Surface catalysis of an L-Ala to D-Ala epimerization process by water molecules adsorbed on to an incipient beta-sheet is suggested as a mechanism whereby crystals of the title peptide were obtained from a solution of tBoc-Pro-D-Ala-Ala-NHCH3.

Structural domains of phytochrome deduced from homologies in amino acid sequences.

A method of semiempirical identification of structural domains is proposed. The procedure is based on the comparison of amino acid sequences in groups of homologous proteins. This approach was tested using 32 known protein sequences from different cytochrome b5, cytochrome c, lysozyme, hemoglobin, and myoglobin proteins. The method presented was able to identify all structural domains of these reference proteins. A consensus secondary structure provided information on structural content of these domains predicting correctly 21 of 23 (91%) of alpha-helices. We applied this method to six homologous phytochrome sequences from Avena, Arabadopsis, Cucurbita, Maize, Oryza, and Pisum. Some of the identified domains can be assigned to the known tertiary structure categories. For example, an alpha/beta domain is localized in the region known to stabilize the phytochrome chromophore in the red light absorbing form (Pr). One alpha-helical and one alpha/beta domains are localized in regions important for the chromophore stabilization in the far-red absorbing form (Pfr). From an analysis of noncovalent interaction patterns in another domain it is proposed that a phytochrome dimer contact involves two segments localized between residues 730 and 821 (using numbering of aligned sequences). Also, a possible antiparallel beta-sheet structure of this region has been suggested. According to this model, the long axis of the interacting structures is perpendicular to a twofold symmetry axis of the phytochrome dimer.

Binding of tachyplesin I to DNA revealed by footprinting analysis: significant contribution of secondary structure to DNA binding and implication for biological action.

In view of the cationic amphipathic structure of tachyplesin I and antiparallel beta-sheet as a general DNA binding motif, DNA binding of the antimicrobial peptide has been examined. Several footprinting-like techniques using DNase I protection, dimethyl sulfate protection, and bleomycin- (BLM-) induced DNA cleavage were applied in this study. Some distinct footprints with DNase I are detected, and also the sequence-specific cleavage mode of the BLM-Fe(II) complex clearly is altered in the presence of tachyplesin I. In addition, methylation of the N-7 residue of guanine situated in the DNA major groove is not entirely inhibited (or activated) by tachyplesin I. The results suggest that tachyplesin I interacts with the minor groove of DNA duplex. Disappearance of the footprints by dithiothreitol-treated tachyplesin I and Ala-tachyplesin strongly suggests a significant contribution of secondary structure containing an antiparallel beta-sheet to the DNA binding of tachyplesin I. This is the first report on DNA interaction with a small peptide which contains a unique antiparallel beta-sheet structure. The mechanism for antimicrobial action of tachyplesin I has also been inferred.

Desmosomal glycoprotein DGI, a component of intercellular desmosome junctions, is related to the cadherin family of cell adhesion molecules.

Among the variety of specialized intercellular junctions, those of the adherens type have the most obvious association with cytoskeletal elements. This may be with the actin microfilament system as in the zonula adherens or with intermediate filaments as in the macula adherens, or desmosome. In the former case, it is clear that transmembrane glycoproteins of the cadherin family are important adhesive components of the molecular assembly. We now show for desmosomes that a major glycoprotein component (desmosomal glycoprotein DGI) has extensive homology with the cadherins, defining an extended family, but also has unique features in its cytoplasmic domain that are likely to be relevant to the association with intermediate rather than actin filaments. A novel 282-residue extension contains repeats of approximately 29 amino acid residues predicted to have an antiparallel beta-sheet structure, followed by a glycine-rich sequence. As in the cadherins, the extracellular domain contains possible Ca2(+)-binding sequences and a potential protease processing site. The cell adhesion recognition region (His-Ala-Val) of the cadherins is modified to Arg-Ala-Leu.

Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: sequence-specific assignments, secondary structure, and dimer formation

The solution structure of neuronal bungarotoxin (nBgt) has been studied by using two-dimensional 1H NMR spectroscopy. Sequence-specific assignments for over 95% of the backbone resonances and 85% of the side-chain resonances have been made by using a series of two-dimensional spectra at four temperatures. From these assignments over 75% of the NOESY spectrum has been assigned, which has in turn provided 582 distance constraints. Twenty-seven coupling constants (NH-alpha CH) were determined from the COSY spectra, which have provided dihedral angle constraints. In addition, hydrogen exchange experiments have suggested the probable position of hydrogen bonds. The NOE constraints, dihedral angle constraints, and the rates of amide proton exchange suggest that a triple-stranded antiparallel beta sheet is the major component of secondary structure, which includes 25% of the amino acid residues. A number of NOE peaks were observed that were inconsistent with the antiparallel beta-sheet structure. Because we have confirmed by sedimentation equilibrium that nBgt exists as a dimer, we have reinterpreted these NOE constraints as intermolecular interactions. These constraints suggest that the dimer consists of a six-stranded antiparallel beta sheet (three from each monomer), with residues 55-59 forming the dimer interface.

Zinc-induced secondary structure transitions in human sperm protamines

Using CD we show that human group II protamines undergo novel zinc-dependent secondary structure transitions. The CD spectra of protamine is characteristic of random coil proteins with a large minima at 197 nm. Upon the addition of 1 mM zinc, the magnitude of this minima is decreased by 44%. This spectral change is not induced by 1 mM calcium or magnesium. Cadmium, which has chemical properties similar to zinc, can also induce the structural transition although not as effectively as zinc. The spectral changes that accompany zinc binding are indicative of an increase in beta-turn and anti-parallel beta-sheet structures. This is consistent with the predicted secondary structure for protamines which is dominated by beta-turns. Our data support a model in which protamine adopts a folded structure in the presence of zinc. We propose that a zinc-modulated structure is physiologically significant considering the relatively high levels of zinc in human sperm.

Determination of the secondary structure and molecular topology of interleukin-1.beta. by use of two- and three-dimensional heteronuclear nitrogen-15-proton NMR spectroscopy

A study of the regular secondary structure elements of recombinant human interleukin-1 beta has been carried out using NMR spectroscopy. Using a randomly 15N labeled sample, a number of heteronuclear three- and two-dimensional NMR experiments have been performed, which have enabled a complete analysis of short-, medium-, and long-range NOEs between protons of the polypeptide backbone, based on the sequence-specific resonance assignments that have been reported previously [Driscoll, P. C., Clore, G. M., Marion, D., Wingfield, P. T., & Gronenborn, A. M. (1990) Biochemistry 29, 3542-3556]. In addition, accurate measurements of a large number of 3JHN alpha coupling constants have been carried out by two-dimensional heteronuclear multiple-quantum-coherence-J spectroscopy. Amide NH solvent exchange rates have been measured by following the time dependence of the 15N-1H correlation spectrum of interleukin-1 beta on dissolving the protein in D2O solution. Analysis of these data indicate that the structure of interleukin-1 beta consists of 12 extended beta-strands aligned in a single extended network of antiparallel beta-sheet structure that in part folds into a skewed six-stranded beta-barrel. In the overall structure the beta-strands are connected by tight turns, short loops, and long loops in a manner that displays approximate pseudo-three-fold symmetry. The secondary structure analysis is discussed in the light of the unrefined X-ray structure of interleukin-1 beta at 3-A resolution [Priestle, J. P., Schär, H.-P., & Grütter, M. G. (1988) EMBO J. 7, 339-343], as well as biological activity data. Discernible differences between the two studies are highlighted. Finally, we have discovered conformational heterogeneity in the structure of interleukin-1 beta, which is characterized by an exchange rate that is slow on the NMR chemical shift time scale.

Molecular determinants of amyloid deposition in Alzheimer's disease: conformational studies of synthetic .beta.-protein fragments

The amyloid beta-protein (1-42) is a major constituent of the abnormal extracellular amyloid plaque that characterizes the brains of victims of Alzheimer's disease. Two peptides, with sequences derived from the previously unexplored C-terminal region of the beta-protein, beta 26-33 (H2N-SNKGAIIG-CO2H) and beta 34-42 (H2N-LMVGGVVIA-CO2H), were synthesized and purified, and their solubility and conformational properties were analyzed. Peptide beta 26-33 was found to be freely soluble in water; however, peptide beta 34-42 was virtually insoluble in aqueous media, including 6 M guanidinium thiocyanate. The peptides formed assemblies having distinct fibrillar morphologies and different dimensions as observed by electron microscopy of negatively stained samples. X-ray diffraction revealed that the peptide conformation in the fibrils was cross-beta. A correlation between solubility and beta-structure formation was inferred from FTIR studies: beta 26-33, when dissolved in water, existed as a random coil, whereas the water-insoluble peptide beta 34-42 possessed antiparallel beta-sheet structure in the solid state. Solubilization of beta 34-42 in organic media resulted in the disappearance of beta-structure. These data suggest that the sequence 34-42, by virtue of its ability to form unusually stable beta-structure, is a major contributor to the insolubility of the beta-protein and may nucleate the formation of the fibrils that constitute amyloid plaque.

Crystal structure and solution conformation of S,S′‐bis(Boc‐Cys‐Ala‐OMe): Intramolecular antiparallel β‐sheet conformation of an acyclic cystine peptide

The conformation of the acyclic biscystine peptide S,S'-bis(Boc-Cys-Ala-OMe) has been studied in the solid state by x-ray diffraction, and in solution by 1H- and 13C-nmr, ir, and CD methods. The peptide molecule has a twofold rotation symmetry and adopts an intramolecular antiparallel beta-sheet structure in the solid state. The two antiparallel extended strands are stabilized by two hydrogen bonds between the Boc CO and Ala NH groups [N...O 2.964 (3) A, O...HN 2.11 (3) A, and NH...O angle 162 (3) degrees]. The disulfide bridge has a right-handed conformation with the torsion angle C beta SSC beta = 95.8 (2) degrees. In solution the presence of a twofold rotation symmetry in the molecule is evident from the 1H- and 13C-nmr spectra. 1H-nmr studies, using solvent and temperature dependencies of NH chemical shifts, paramagnetic radical induced line broadening, and rate of deuterium-hydrogen exchange effects on NH resonances, suggest that Ala NH is solvent shielded and intramolecularly hydrogen bonded in CDCl3 and in (CD3)2SO. Nuclear Overhauser effects observed between Cys C alpha H and Ala NH protons and ir studies provide evidence of the occurrence of antiparallel beta-sheet structure in these solvents. The CD spectra of the peptide in organic solvents are characteristic of those observed for cystine peptides that have been shown to adopt antiparallel beta-sheet structures.

A Histidine Residue at the Active Site of Avian Liver Phosphoenolpyruvate Carboxykinase

The histidine-selective reagents diethylpyrocarbonate (DEPC) and dimethylpyrocarbonate were used to study active site residues of phosphoenolpyruvate carboxykinase. Both reagents show pseudo first-order inhibition of enzyme activity at 22 +/- 1 degree C with calculated second-order rate constants of 2.8 and 4.6 M-1 s-1, respectively. The inhibition appears partially reversible. Substrates affect the rate of inhibition: KHCO3 enhances the rate, Mn2+ has little effect, and phosphoenolpyruvate decreases the rate. The best protection is obtained by IDP or IDP and Mn2+. The kinetic studies show that modification of histidine is specific and leads to loss of enzymatic activity. Two histidines per enzyme are modified by DEPC, as measured by an absorption change at 240 nm, in the absence of substrate, leading to loss in activity. One histidine per molecule is modified in the presence of KHCO3, giving inactivation. Cysteine and lysine residues are not affected. A study of the inhibition rate constant as a function of pH gives a pKa of 6.7. Enzyme modified by DEPC in the absence of substrate (1% remaining activity) shows no binding of ITP or of phosphoenolpyruvate to the enzyme.Mn2+ complex as studied by proton relaxation rates. When enzyme is modified in the presence of KHCO3 (44% remaining activity), ITP and KHCO3 bind to the enzyme.Mn2+ complex similarly to the binding to native enzyme. Phosphoenolpyruvate binding to modified enzyme.Mn results in an enhancement of proton relaxation rates rather than the decrease observed with native enzyme.Mn. The CD spectra of histidine-modified enzyme show a decrease in alpha-helical and random structure with an increase in anti-parallel beta-sheet structure compared to native enzyme. These results show that avian phosphoenolpyruvate carboxykinase has 2 histidine residues which are reactive with DEPC and dimethylpyrocarbonate, and one of the 15 histidine residues in the protein is at or near the phosphoenolpyruvate binding site and is involved in catalysis.

Structural characterization of the interactions between calmodulin and skeletal muscle myosin light chain kinase: effect of peptide (576-594)G binding on the Ca2+-binding domains.

Calcium-containing calmodulin (CaM) and its complex with a peptide corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase [skMLCK(576-594)G] have been studied by one- and two-dimensional 1H NMR techniques. Resonances arising from the antiparallel beta-sheet structures associated with the calcium-binding domains of CaM and their counterparts in the CaM-skMLCK(576-594)G complex have been assigned. The assignments were initiated by application of the main chain directed assignment strategy. It is found that, despite significant changes in chemical shifts of resonances arising from amino acid residues in this region upon binding of the peptide, the beta-sheets have virtually the same structure in the complex as in CaM. Hydrogen exchange rates of amide NH within the beta-sheet structures are significantly slowed upon binding of peptide. These data, in conjunction with the observed nuclear Overhauser effect (NOE) patterns and relative intensities and the downfield shifts of associated amide and alpha resonances upon binding of peptide, show that the peptide stabilizes the Ca2+-bound state of calmodulin. The observed pattern of NOEs within the beta-sheets and their structural similarity correspond closely to those predicted by the crystal structure. These findings imply that the apparent inconsistency of the crystal structure with recently reported low-angle X-ray scattering profiles of CaM may lie within the putative central helix bridging the globular domains.

Sequence-specific 1H-n.m.r. assignments and peptide backbone conformation in rat epidermal growth factor.

The solution conformation of rat epidermal growth factor (EGF) has been investigated by proton n.m.r. techniques. Two-dimensional proton n.m.r. experiments have allowed sequential resonance assignments to be made for most protons. On the basis of these assignments, two regions of anti-parallel beta-sheet structure have been derived from the n.m.r. data. A beta-sheet segment running from about V19 to V23 (capital letters refer to amino acids in the single-letter notation) is folded onto a beta-sheet segment running from R28 to N32 and joined by a chain reversal from E24 to D27. A second region involves a beta-turn from V34 to Y37, which starts a short beta-sheet up to G39, followed by a chain reversal up to Q43, which leads to folding of the C-terminal beta-sheet segment, i.e. H44-R45, running antiparallel to the short Y37 beta-sheet segment. The N-terminal segment up to G18 exists in a multiple bend conformation and is folded on to the V29-V23/R28-N32 beta-sheet such that Y10, Y13, Y22 and Y29 are proximal to each other. Structural comparison of rat, murine and human EGFs indicates a number of highly conserved structural features common to at least these species of EGF.

Lipid-induced changes in the secondary structure of snake venom cardiotoxins.

The secondary structures of three snake venom cardiotoxins (from Hemachatus hemachatus, Naja naja atra, and Naja naja naja), in aqueous solution and in a lipid-bound form, were investigated by Fourier-transform infrared spectroscopy. The conformation-sensitive protein infrared bands in the amide I region were analyzed using deconvolution and band-fitting procedures. The spectra of the three cardiotoxins in aqueous buffer are very similar; they indicate a high content of both antiparallel beta-sheet structure and unordered conformation. Moreover, component bands characteristic of turns can also be identified. The binding of cardiotoxins to bilayers of dimyristoylphosphatidyl-glycerol results in an increased content of a beta-structure at the expense of the nonordered conformation. It is suggested that lipid-induced conformational transitions to a beta-structure, similar to that observed with cardiotoxins, may be operative also in membrane interaction of other proteins and peptides, particularly with those which have a small tendency to form alpha-helices.

Prediction of Secondary Structure, Spatial Organization and Distribution of Antigenic Determinants for Hepatitis A Virus Proteins

On the basis of the secondary structure calculations from the known amino acid sequence we came to the conclusion that hepatitis A virus capsid proteins have the typical antiparallel beta-sheet bilayer structure. The predicted secondary structure of the HAV proteins can be well aligned with those of the poliovirus (type 1 Mahoney) and human rhinovirus (type 14). It enabled us to use the X-ray structure of the PV-1M and HRV-14 proteins as a template and then, firstly, to localize the positions of alpha and beta regions in the architecture of the HAV protein molecules and, secondly, to discover the amino acid homologies of the secondary structure regions aligned. The obtained model of the three-dimensional structure for HAV proteins helped us to indicate the exposed regions of the polypeptide chains and to pinpoint the potential neutralizing antigenic sites.