Steroidal constituents of rice (Rryza sativa) hulls with algicidal and herbicidal activity against blue-green algae and duckweed

Two new compounds, 14-methyl stigmast-9(11)-en-3alpha-ol-3beta-D-glucopyranoside (1) and cholest-11-en-3beta, 6beta, 7alpha, 22beta-tetraol-24-one-3beta-palmitoleate (2), along with the known compound beta-sitosteryl-3beta-D-glucopyranosyl-6'-linoleiate (3), were isolated from the methanolic extract of rice (Oryza sativa) hulls. The structures of the two new compounds were elucidated using one- and two-dimensional NMR in combination with IR, EI/MS, FAB/MS, HR-EI/MS and HR-FAB/MS. In bioassays with blue-green algae, Microcystis aeruginosa UTEX 2388 and duckweed, Lemna paucicostata Hegelm 381, the efficacy of bioactivity of the two new compounds linearly increased as the concentration increased from 0.3 to 300 IgM. Compared with momilactone A, compounds 1 and 2 showed similar and higher inhibitory activities against the growth of M. aeruginosa at a concentration of 300 microM. However, compound 2 was similar to momilactone A in inhibiting L. paucicostata growth at a concentration of 300 microM. As a result, compound 2 appears to have a strong potential for the environmentally friendly control of weed and algae that are harmful to water-logged rice.     

Minimal surface as a model of beta-sheets

The purpose of this article is to present arguments based on experimental data that the beta-sheet structures in proteins are the result of the tendency to minimize surface areas. Thus, we propose the model that all beta-sheet structures are almost minimal surfaces, namely, their mean curvatures are nearly zero. To support this model, we chose 1740 disjoint beta-sheets with less than 10 strands from the all beta-protein class in a nonredundant 40% Structural Classification of Proteins (SCOP) database and applied the least-squares method to fit the minimal surface catenoid (and in some rare cases, the plane) to the beta-sheet structures. The fitting errors were extremely small: The error of 1729 beta-sheets with catenoid minimal surface is 0.90 +/- 0.55 A and the error of the remaining 11 flat sheets with the plane is 0.64 +/- 0.46 A. The fact that the commonly used models for some beta-sheet surfaces (i.e., the hyperboloid and strophoid) have very small mean curvatures (< 0.05) supports our model. Moreover, we showed that this model also includes the isotropically stressed configuration model proposed by Salemme, in which the intrastrand tendency of the individual chains to twist or coil is in equilibrium with the tendency of the interstrand hydrogen bonding to resist twisting of the sheet as a whole. As an application we used our model to quantify the two principal independent modes in the flexibility of beta-sheets, that is, the bending parameter of beta-sheets and the inclined angle of beta-strands in a sheet.     

Biomolecular structures of naturally occurring carbohydrate polymers

The bonding mechanism in proteins and polysaccharides is compared and contrasted. Some of the structures for polyglucose are shown to mimic the classic β-sheet, -helix and collagen helices found in proteins. The effects of glycosidic linkage geometry on polysaccharide shape and structure is discussed together with the effect of changes in the geometry of the monomer units. Multistranded ropes and the effect of non-carbohydrate groups are mentioned briefly.     

Chemical constituents of rice (Oryza sativa) hulls and their herbicidal activity against duckweed (Lemna paucicostata Hegelm 381)

Four new compounds, stigmastanol-3beta-p-glyceroxydihydrocoumaroate (1), stigmastanol-3beta-p-butanoxydihydrocoumaroate (2), lanast-7,9(11)-dien-3alpha,15alpha-diol-3alpha-D-glucofuranoside (3) and 1-phenyl-2-hydroxy-3,7-dimethyl-11-aldehydic-tetradecane-2-beta-D-glucopyranoside (4), along with several known compounds were isolated from the methanol extract of hulls of Oryza sativa. The new structures were established by one- and two-dimensional NMR and in combination with IR, EI/MS, FAB/MS and HR-FAB/ MS. Compound (3) strongly inhibited the growth of duckweed (Lemna paucicostata Hegelm 381), whilst compounds (2) and (4) exhibited weak inhibition.     

Direct folding simulation of alpha-helices and beta-hairpins based on a single all-atom force field with an implicit solvation model

Recently, we have shown that a modified energy model based on the param99 force field with the generalized Born (GB) solvation model produces reliable free energy landscapes of mini-proteins with a betabetaalpha motif (BBA5, 1FSD, and 1PSV), with the native structures of the mini-proteins located in their lowest free energy minimum states. One of the main features in the modified energy model is a significant improvement for more balanced treatments of alpha and beta strands in proteins. In this study, using the replica exchange molecular dynamics (REMD) simulation method with this new force field, we have carried out extensive ab initio folding studies of several well-known peptides with alpha or beta strands (C-peptide, EK-peptide, le0q, and gbl). Starting from fully extended conformations as the initial conditions, all of the native-like structures of the target peptides were successfully identified by REMD, with reasonable representations of free energy surfaces. The present simulation results with the modified energy model are consistent with experiments, demonstrating an extended applicability of the energy model to folding studies of a variety of alpha-helices, beta-strands, and alpha/beta proteins.     

Analysis of interactive packing of secondary structural elements in alpha/beta units in proteins

An alpha-helix and a beta-strand are said to be interactively packed if at least one residue in each of the secondary structural elements loses 10% of its solvent accessible contact area on association with the other secondary structural element. An analysis of all such 5,975 nonidentical alpha/beta units in protein structures, defined at < or = 2.5 A resolution, shows that the interaxial distance between the alpha-helix and the beta-strand is linearly correlated with the residue-dependent function, log[(V/nda)/n-int], where V is the volume of amino acid residues in the packing interface, nda is the normalized difference in solvent accessible contact area of the residues in packed and unpacked secondary structural elements, and n-int is the number of residues in the packing interface. The beta-sheet unit (beta u), defined as a pair of adjacent parallel or antiparallel hydrogen-bonded beta-strands, packing with an alpha-helix shows a better correlation between the interaxial distance and log(V/nda) for the residues in the packing interface. This packing relationship is shown to be useful in the prediction of interaxial distances in alpha/beta units using the interacting residue information of equivalent alpha/beta units of homologous proteins. It is, therefore, of value in comparative modeling of protein structures.     

Computational analysis of glycoside hydrolase family 1 specificities

Glycoside hydrolase family 1 consists of beta-glucosidases, beta-galactosidases, 6-phospho-beta-galactosidases, myrosinases, and other enzymes having similar primary and tertiary structures but diverse specificities. Among these enzymes, beta-glucosidases hydrolyze cellobiose to glucose, and therefore they are key players in any cellulose to glucose process. All family members attack beta-glycosidic bonds between a pyranosyl glycon and an aglycon, but most have little specificity for the aglycon or for the bond configuration. Furthermore, glycon specificity is not absolute. Sixteen family members (six beta-glucosidases, two cyanogenic beta-glucosidases, one 6-phospho-beta-galactosidase, two myrosinases, and five beta-glycosidases) have known tertiary structures. We have used automated docking to computationally bind disaccharides with allopyranosyl, galactopyranosyl, glucopyranosyl, mannopyranosyl, 6-phosphogalactopyranosyl, and 6-phosphoglucopyranosyl glycons, all linked by beta-(1,2), beta-(1,3), beta-(1,4), and beta-(1,6)-glycosidic bonds to beta-glucopyranoside aglycons, along with beta-(1,1-thio)-allopyranosyl, -galactopyranosyl, -glucopyranosyl, and -mannopyranosyl) beta-glucopyranosides, into all of these structures to investigate the structural determinants of their enzyme specificities. The following are the eight active-site residues: Glu191, Thr194, Phe205, Asn285, Arg336, Asn376, Trp378, and Trp465 (Zea mays beta-glucosidase numbering), that control a significant amount of glycon specificity. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1021-1031, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Regulation of the Type II Hemidesmosomal Plaque Assembly in Intestinal Epithelial Cells

Hemidesmosomes (HDs) are cellular junctions that anchor epithelial cells to the extracellular matrix (ECM) and are associated morphologically with the cytoskeleton. Hemidesmosomal molecular components include two proteins involved in linking intermediate filaments, HD1/plectin and BP230, and two transmembrane proteins, BP180 and the alpha6beta4 integrin, a laminin receptor. In cells lacking BP230 and BP180, HD1/plectin still associates with alpha6beta4 integrin, forming HD-like structures, called type II HDs. In the present study, we used an intestinal epithelial cell line that expresses HD1/plectin and the alpha6beta4 integrin to investigate the regulation of assembly of these proteins in type II HDs. These compounds were found to be clustered at sites of cell-ECM contact and their polarized localization was influenced by either cell confluency or extracellular matrix deposition. Conventional and immunoelectron microscopy showed that HD1/plectin and the beta4 integrin subunit are colocalized in an adhesion structure. Using cytoskeleton-disrupting drugs and confocal microscopy, we demonstrated that type II HDs are made up of numerous individual plaques whose assembly into a cluster requires actin filaments, but not microtubules.     

Reinvestigation of the proposed folding and self-association of the Neuropeptide Head Activator

The Neuropeptide Head Activator (HA), pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe (pGlu is pyroglutamic acid), is involved in head-specific growth and differentiation processes in the freshwater coelenterate Hydra attenuata. Peptides of identical sequence have also been isolated from higher-organism tissues such as human and bovine hypothalamus. Early studies by molecular sieve chromatography suggested that HA dimerizes with high affinity (K(d) approximately 1 nM). This dimerization was proposed to occur via antiparallel beta-sheet formation between the Lys(7)-Phe(11) segments in each HA molecule. We conducted biophysical studies on synthetic HA in order to gain insight into its structure and aggregation tendencies. We found by analytical ultracentrifugation that HA is monomeric at low millimolar concentrations. Studies by (1)H-NMR revealed that HA did not adopt any significant secondary structure in solution. We found no NOEs that would support the proposed dimer structure. We probed the propensity of the Lys(7)-Phe(11) fragment to form antiparallel beta-sheet by designing peptides in which two such fragments are joined by a two-residue linker. These peptides were intended to form stable beta-hairpin structures with cross-strand interactions that mimic those of the proposed HA dimer interface. We found that the HA-derived fragments may be induced to form intramolecular beta-sheet, albeit only weakly, when linked by the highly beta-hairpin-promoting D-Pro-Gly turn, but not when linked by the more flexible Gly-Gly unit. These findings suggest that the postulated mode of HA dimerization and the proposed propensity of the molecule to form discrete aggregates with high affinity are incorrect.     

Crystal-state conformation of Calpha,alpha-dialkylated peptides containing chiral beta-homo-residues.

Secondary structure formation and stability are essential features in the knowledge of complex folding topology of biomolecules. To better understand the relationships between preferred conformations and functional properties of beta-homo-amino acids, the synthesis and conformational characterization by X-ray diffraction analysis of peptides containing conformationally constrained Calpha,alpha-dialkylated amino acid residues, such as alpha-aminoisobutyric acid or 1-aminocyclohexane-1-carboxylic acid and a single beta-homoamino acid, differently displaced along the peptide sequence have been carried out. The peptides investigated are: Boc-betaHLeu-(Ac6c)2-OMe, Boc-Ac6c-betaHLeu-(Ac6c)2-OMe and Boc-betaHVal-(Aib)5-OtBu, together with the C-protected beta-homo-residue HCl.H-betaHVal-OMe. The results indicate that the insertion of a betaH-residue at position 1 or 2 of peptides containing strong helix-inducing, bulky Calpha,alpha-disubstituted amino acid residues does not induce any specific conformational preferences. In the crystal state, most of the NH groups of beta-homo residues of tri- and tetrapeptides are not involved in intramolecular hydrogen bonds, thus failing to achieve helical structures similar to those of peptides exclusively constituted of Calpha,alpha-disubstituted amino acid residues. However, by repeating the structural motifs observed in the molecules investigated, a beta-pleated sheet secondary structure, and a new helical structure, named (14/15)-helix, were generated, corresponding to calculated minimum-energy conformations. Our findings, as well as literature data, strongly indicate that conformations of betaH-residues, with the micro torsion angle equal to -60 degrees, are very unlikely.     

Conformational investigation of alpha,beta-dehydropeptides. XVI. Beta-turn tendency in Ac-Pro-DeltaXaa-NHMe: crystallographic and theoretical studies.

The crystal structures of two diastereomeric alpha,beta-dehydrobutyrine peptides Ac-Pro-(Z)-DeltaAbu-NHMe (I) and Ac-Pro-(E)-DeltaAbu-NHMe (II) have been determined. Both dehydropeptides adopt betaI-turn conformation characterized by the pairs of (phi(i+1), psi(i+1)) and (phi(i+2), psi(i+2)) angles as -66, -19, -97, 11 degrees for I and -59, -27, -119, 29 degrees for II. In each peptide, the betaI turn is stabilized by (i + 3) --> i intramolecular hydrogen bonds with N...O distance of 3.12 A for I and 2.93 A for II. These structures have been compared to the crystal structures of homologous peptides Ac-Pro-DeltaVal-NHMe and Ac-Pro-DeltaAla-NHMe. Theoretical analyses by DFT/B3LYP/6-31 + G** method of conformers formed by these four peptides and by the saturated peptide Ac-Pro-Ala-NHMe revealed that peptides with a (Z) substituent at the C(beta) (i+2) atom of dehydroamino acid, i.e. Ac-Pro-DeltaVal-NHMe and Ac-Pro-(Z)-DeltaAbu-NHMe, predominantly form beta turns, both in vacuo and in polar environment. The tendency to adopt beta-turn conformation is much weaker for the peptides lacking the (Z) substituent, Ac-Pro-(E)-DeltaAbu-NHMe and Ac-Pro-DeltaAla-NHMe. The latter adopts a semi-extended or an extended conformation in every polar environment, including a weakly polar solvent. The saturated peptide Ac-Pro-Ala-NHMe in vacuo prefers a beta-turn conformation, but in polar environment the differences between various conformers are small. The role of pi-electron correlation and intramolecular hydrogen bonds interaction in stabilizing the hairpin structures are discussed.     

Mechanisms of action of corilagin and tellimagrandin I that remarkably potentiate the activity of beta-lactams against methicillin-resistant Staphylococcus aureus

Corilagin and tellimagrandin I are polyphenols isolated from the extract of Arctostaphylos uvaursi and Rosa canina L. (rose red), respectively. We have reported that corilagin and tellimagrandin I remarkably reduced the minimum inhibitory concentration (MIC) of beta-lactams in methicillin-resistant Staphylococcus aureus(MRSA). In this study, we investigated the effect of corilagin and tellimagrandin I on the penicillin binding protein 2 '(2a) (PBP2 '(PBP2a)) which mainly confers the resistance to beta-lactam antibiotics in MRSA. These compounds when added to the culture medium were found to decrease production of the PBP2 '(PBP2a) slightly. Using BOCILLIN FL, a fluorescent-labeled benzyl penicillin, we found that PBP2 '(PBP2a) in MRSA cells that were grown in medium containing corilagin or tellimagrandin I almost completely lost the ability to bind BOCILLIN FL. The binding activity of PBP2 and PBP3 were also reduced to some extent by these compounds. These results indicate that inactivation of PBPs, especially of PBP2 '(PBP2a), by corilagin or tellimagrandin I is the major reason for the remarkable reduction in the resistance level of beta-lactams in MRSA. Corilagin or tellimagrandin I suppressed the activity of beta-lactamase to some extent.     

Pyrazolopyrimidine metabolism in Leishmania and trypanosomes: significant differences between host and parasite

The pathogenic hemoflagellates of the genera Leishmania and Trypanosoma are major causes of human disease in the tropical and subtropical areas of the world. In general, the agents used to treat diseases caused by these organisms are toxic and not suitable for administration to the millions of people infected. Investigations over the past several years have shown that there are several major differences between man and these protozoans with respect to purine metabolism. The differences appear to offer promise for the development of effective chemotherapeutic compounds. These organisms do not synthesize purines de novo, as does man. They are able to concentrate pyrazolopyrimidines with the cell and metabolize them as purines through the salvage pathways, ultimately incorporating them into nucleic acids. This does not occur in mammals. The pyrazolopyrimidine base allopurinol, which has served as a prototype, is activated by a phosphoribosyltransferase to the ribonucleotide. The ribonucleotide is aminated to the 4-amino-pyrazolopyrimidine ribonucleotide and subsequently phosphorylated to the triphosphate form and incorporated into RNA. The pyrazolopyrimidine ribonucleosides formycin B and allopurinol ribonucleoside are activated through a nucleoside phosphotransferase. The resulting ribonucleotide is aminated and incorporated into RNA as described above. These metabolic peculiarities occur not only in the forms of these parasites which are found in the insect vectors but also in the intracellular forms which are pathogenic in man. The differences in the enzymology and metabolism of purines which exist in the genera Leishmania and Trypanosoma offer excellent opportunities for chemotherapeutic exploitation.     

Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short‐chain acyl esters (C2–C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4‐nitrophenyl‐β‐D ‐xylopyranoside monoacetates as substrates in a β‐xylosidase‐coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine‐substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic α/β‐hydrolase fold. TM0077 assembles into a doughnut‐shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Computational studies and peptidomimetic design for the human p53-MDM2 complex

The interaction between human p53 and MDM2 is a key event in controlling cell growth. Many studies have suggested that a p53 mimic would be sufficient to inhibit MDM2 to reduce cell growth in cancerous tissue. In order to design a potent p53 mimic, molecular dynamics (MD) simulations were used to examine the binding interface and the effect of mutating key residues in the human p53-MDM2 complex. The Generalized Born surface area (GBSA) method was used to estimate free energies of binding, and a computational alanine-scanning approach was used to calculate the relative effects in the free energy of binding for key mutations. Our calculations determine the free energy of binding for a model p53-MDM2 complex to be -7.4 kcal/mol, which is in very good agreement with the experimentally determined values (-6.6--8.8 kcal/mol). The alanine-scanning results are in good agreement with experimental data and calculations by other groups. We have used the information from our studies of human p53-MDM2 to design a beta-peptide mimic of p53. MD simulations of the mimic bound to MDM2 estimate a free energy of binding of -8.8 kcal/mol. We have also applied alanine scanning to the mimic-MDM2 complex and reveal which mutations are most likely to alter the binding affinity, possibly giving rise to escape mutants. The mimic was compared to nutlins, a new class of inhibitors that block the formation of the p53-MDM2 complex. There are interesting similarities between the nutlins and our mimic, and the differences point to ways that both inhibitors may be improved. Finally, an additional hydrophobic pocket is noted in the interior of MDM2. It may be possible to design new inhibitors to take advantage of that pocket.     

Analysis of hydrophobicity in the alpha and beta chemokine families and its relevance to dimerization.

The chemokine family of chemotactic cytokines plays a key role in orchestrating the immune response. The family has been divided into 2 subfamilies, alpha and beta, based on the spacing of the first 2 cysteine residues, function, and chromosomal location. Members within each subfamily have 25-70% sequence identity, whereas the amino acid identity between members of the 2 subfamilies ranges from 20 to 40%. A quantitative analysis of the hydrophobic properties of 11 alpha and 9 beta chemokine sequences, based on the coordinates of the prototypic alpha and beta chemokines, interleukin-8 (IL-8), and human macrophage inflammatory protein-1 beta (hMIP-1 beta), respectively, is presented. The monomers of the alpha and beta chemokines have their strongest core hydrophobic cluster at equivalent positions, consistent with their similar tertiary structures. In contrast, the pattern of monomer surface hydrophobicity between the alpha and beta chemokines differs in a manner that is fully consistent with the observed differences in quaternary structure. The most hydrophobic surface clusters on the monomer subunits are located in very different regions of the alpha and beta chemokines and comprise in each case the amino acids that are buried at the interface of their respective dimers. The theoretical analysis of hydrophobicity strongly supports the hypothesis that the distinct dimers observed for IL-8 and hMIP-1 beta are preserved for all the alpha and beta chemokines, respectively. This provides a rational explanation for the lack of receptor crossbinding and reactivity between the alpha and beta chemokine subfamilies.     

Side-chain entropy effects on protein secondary structure formation

Loss of conformational entropy is one of the primary factors opposing protein folding. Both the backbone and side-chain of each residue in a protein will have their freedom of motion restricted in the final folded structure. The type of secondary structure of which a residue is part will have a significant impact on how much side-chain entropy is lost. Side-chain conformational entropies have previously been determined for folded proteins, simple models of unfolded proteins, alpha-helices, and a dipeptide model for beta-strands, but not for polyproline II (PII) helices. In this work, we present side-chain conformational estimates for the three regular secondary structure types: alpha-helices, beta-strands, and PII helices. Entropies are estimated from Monte Carlo computer simulations. Beta-strands are modeled as two structures, parallel and antiparallel beta-strands. Our data indicate that restraining a residue to the PII helix or antiparallel beta-strand conformations results in side-chain entropies equal to or higher than those obtained by restraining residues to the parallel beta-strand conformation. Side-chains in the alpha-helix conformation have the lowest side-chain entropies. The observation that extended structures retain the most side-chain entropy suggests that such structures would be entropically favored in unfolded proteins under folding conditions. Our data indicate that the PII helix conformation would be somewhat favored over beta-strand conformations, with antiparallel beta-strand favored over parallel. Notably, our data imply that, under some circumstances, residues may gain side-chain entropy upon folding. Implications of our findings for protein folding and unfolded states are discussed.     

Recent advances in understanding the mechanism of hemozoin (malaria pigment) formation

The recent literature on hemozoin/β-hematin formation is reviewed, with an emphasis on the mechanism of its formation. Recent findings from unrelated organisms that produce hemozoin, namely the malaria parasite Plasmodium falciparum, the worm Schistosoma mansoni and the kissing bug Rhodnius prolixus all of which consume human hemoglobin show that the formation of this crystalline substance occurs within or at the surface of lipids. Biomimetic experimental models of the lipid–water interface as well as computational studies indicate that these lipid environments are probably extraordinarily efficient at producing hemozoin. A rethink is now needed, with a new emphasis on Fe(III)PPIX in non-aqueous environments that mimic lipids and indeed within the lipid environment itself. These findings are explored and discussed in the context of earlier studies on β-hematin formation.     

Autonomous folding of a peptide corresponding to the N-terminal beta-hairpin from ubiquitin.

The N-terminal 17 residues of ubiquitin have been shown by 1H NMR to fold autonomously into a beta-hairpin structure in aqueous solution. This structure has a specific, native-like register, though side-chain contacts differ in detail from those observed in the intact protein. An autonomously folding hairpin has previously been identified in the case of streptococcal protein G, which is structurally homologous with ubiquitin, but remarkably, the two are not in topologically equivalent positions in the fold. This suggests that the organization of folding may be quite different for proteins sharing similar tertiary structures. Two smaller peptides have also been studied, corresponding to the isolated arms of the N-terminal hairpin of ubiquitin, and significant differences from simple random coil predictions observed in the spectra of these subfragments, suggestive of significant limitation of the backbone conformational space sampled, presumably as a consequence of the strongly beta-structure favoring composition of the sequences. This illustrates the ability of local sequence elements to express a propensity for beta-structure even in the absence of actual sheet formation. Attempts were made to estimate the population of the folded state of the hairpin, in terms of a simple two-state folding model. Using published "random coil" values to model the unfolded state, and values derived from native ubiquitin for the putative unique, folded state, it was found that the apparent population varied widely for different residues and with different NMR parameters. Use of the spectra of the subfragment peptides to provide a more realistic model of the unfolded state led to better agreement in the estimates that could be obtained from chemical shift and coupling constant measurements, while making it clear that some other approaches to population estimation could not give meaningful results, because of the tendency to populate the beta-region of conformational space even in the absence of the hairpin structure.     

Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy.

Pathogenic prion proteins (PrP(Sc)) are thought to be produced by alpha-helical to beta-sheet conformational changes in the normal cellular prion proteins (PrP(C)) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129-154) and PrP(192-213); the former is supposed to assume beta-sheets and the latter alpha-helices, in PrP(Sc). The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129-154) and PrP(192-213) mainly adopted random-coils (approximately 60%), followed by beta-sheets (30%-40%). PrP(129-154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-beta-D-glucopyranoside (OG), octy-beta-D-maltopyranoside (OM). sodium dodecyl sulfate (SDS), Zwittergent 3-14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192-213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the alpha-helical content, and decreased the beta-sheet and random-coil contents. DPC also increased the alpha-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129-154) has a propensity to adopt predominantly beta-sheets. On the other hand, PrP(192-213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrP(C) could be converted into a nascent PrP(Sc) having a transient PrP(Sc) like structureunder the hydrophobic environments produced by gangliosides.