Pulmonary surfactant-associated polypeptide C in a mixed organic solvent transforms from a monomeric alpha-helical state into insoluble beta-sheet aggregates.

In the 35-residue pulmonary surfactant-associated lipopolypeptide C (SP-C), the stability of the valyl-rich alpha-helix comprising residues 9-34 has been monitored by circular dichroism, nuclear magnetic resonance, and Fourier transform infrared spectroscopy in both a mixed organic solvent and in phospholipid micelles. The alpha-helical form of SP-C observed in freshly prepared solutions in a mixed solvent of CHCl3/CH3OH/0.1 M HCl 32:64:5 (v/v/v) at 10 degrees C undergoes within a few days an irreversible transformation to an insoluble aggregate that contains beta-sheet secondary structure. Hydrogen exchange experiments revealed that this conformational transition proceeds through a transition state with an Eyring free activation enthalpy of about 100 kJ mol(-1), in which the polypeptide segment 9-27 largely retains a helical conformation. In dodecylphosphocholine micelles, the helical form of SP-C was maintained after seven weeks at 50 degrees C. The alpha-helical form of SP-C thus seems to be the thermodynamically most stable state in this micellar environment, whereas its presence in freshly prepared samples in the aforementioned mixed solvent is due to a high kinetic barrier for unfolding. These observations support a previously proposed pathway for in vivo synthesis of SP-C through proteolytic processing from a 21-kDa precursor protein.     

Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy

A simple approach to estimate the number of alpha-helical and beta-strand segments from protein circular dichroism spectra is described. The alpha-helix and beta-sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given alpha-helix or beta-strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per alpha-helix and two residues per beta-strand may be considered distorted in proteins. The number of alpha-helical and beta-strand segments and their average length in a given protein were estimated from the fraction of distorted alpha-helix and beta-strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.     

Oligopeptide-mediated helix stabilization of model peptides in aqueous solution.

Oligopeptide-mediated helix stabilization of peptides in hydrophobic solutions was previously found by NMR and CD spectroscopic studies. The oligopeptide included the hydrophobic amino acids found in its parent peptide and were interposed by relevant basic oracidic amino acids. The strength of the interactions depended on the amino acid sequences. However, no helix-stabilizing effect was seen for the peptides in phosphate buffer solution, because the peptides assumed a random-coil structure. In order to ascertain whether the helix-stabilizing effect of an oligopeptide on its parent peptide could operate in aqueous solution, model peptides EK17 (Ac-AEAAAAEAAAKAAAAKA-NH2) and IFM17 (Ac-AEAAAAEIFMKAAAAKA-NH2) that may assume an alpha-helix in aqueous solutions were synthesized. Interactions were examined between various oligopeptides (EAAAK, KAAAE, EIFMK, KIFME, KIFMK and EYYEE) and EK17 or IFM17 in phosphate buffer and in 80% trifluoroethanol (TFE)-20% H2O solutions by CD spectra. EAAAK had little effect on the secondary structures of EK17 in both buffer and TFE solutions, while KAAAE, which has the reverse amino acid sequence of EAAAK, had a marked helix-destabilizing effect on EK17 in TFE. EIFMK and KIFME were found to stabilize the alpha-helical structure of EK17 in phosphate buffer solutions, whereas KIFMK and EYYEE destabilized the alpha-helical structure of EK17. EIFMK and KIFME had no effect on IFM17, because unexpectedly, IFM17 had appreciable amounts of beta-sheet structure in buffer solution. It was concluded that in order for the helix-stabilizing (1) the model peptide, the alpha-helical conformation of which is to be stabilized, should essentially assume an alpha-helical structure by nature, and (2) the hydrophobicity of the side-chains of the oligopeptide should be high enough for the oligopeptide to perform stable specific side chain-side chain intermolecular hydrophobic interactions with the model peptide.     

Pressure dependence of human fibrinogen correlated to the conformational alpha-helix to beta-sheet transition: an Fourier transform infrared study microspectroscopic study

We used Fourier transform infrared (FTIR) microspectroscopy to investigate pressure-induced conformational changes in secondary structure of fibrinogen (FBG). Solid state FBG was compressed on a KBr pellet (1KBr method) or between two KBr pellets (2KBr method). The peak positions of the original and second-derivative ir spectra of compressed FBG samples prepared by the 1KBr method were similar to FBG sample without pressure. When FBG was prepared by the 2KBr method and pressure was increased up to 400 kg/cm(2), peaks at 1625 (intermolecular beta-sheet) and 1611 (beta-sheet aggregates structure and/or the side-chain absorption of the tyrosine residues) cm(-1) were enhanced. The peaks near 1661 (beta-sheet) and 1652 (alpha-helix) cm(-1) also exhibited a marked change with pressure. A linear correlation was found between the peak intensity ratio of 1611/1652 cm(-1) (r = 0.9879) or 1625/1652 cm(-1) (r = 0.9752) and applied pressure. The curve-fitted compositional changes in secondary structure of FBG also indicate that the composition of the alpha-helix structure (1657-1659 cm(-1)) was gradually reduced with the increase in compression pressure, but the composition of the beta-sheet structure (1681, 1629, and 1609 cm(-1)) gradually increased. This indicates that pressure-induced conformational changes in FBG include not only transformations from alpha-helix to beta-sheet structure, but also unfolding and denaturation of FBG and the formation of aggregates.     

Free energy surfaces of beta-hairpin and alpha-helical peptides generated by replica exchange molecular dynamics with the AGBNP implicit solvent model

We have studied the potential of mean force of two peptides, one known to adopt a beta-hairpin and the other an alpha-helical conformation in solution. These peptides are, respectively, residues 41-56 of the C-terminus (GEWTYDDATKTFTVTE) of the B1 domain of protein G and the 13 residue C-peptide (KETAAAKFERQHM) of ribonuclease A. Extensive canonical ensemble sampling has been performed using a parallel replica exchange method. The effective potential employed in this work consists of the OPLS all-atom force field (OPLS-AA) and an analytical generalized Born (AGB) implicit solvent model including a novel nonpolar solvation free energy estimator (NP). An additional dielectric screening parameter has been incorporated into the AGBNP model. In the case of the beta-hairpin, the nonpolar solvation free energy estimator provides the necessary effective interactions for the collapse of the hydrophobic core (W43, Y45, F52, and V54), which the more commonly used surface-area-dependent nonpolar model does not provide. For both the beta-hairpin and the alpha-helix, increased dielectric screening reduces the stability of incorrectly formed salt bridges, which tend to disrupt the formation of the hairpin and helix, respectively. The fraction of beta-hairpin and alpha-helix content we obtained using the AGBNP model agrees well with experimental results. The thermodynamic stability of the beta-hairpin from protein G and the alpha-helical C-peptide from ribonuclease A as modeled with the OPLS-AA/AGBNP effective potential reflects the balance between the nonpolar effective potential terms, which drive compaction, and the polar and hydrogen bonding terms, which promote secondary structure formation.     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

Structural transformation and aggregation of human alpha-synuclein in trifluoroethanol: non-amyloid component sequence is essential and beta-sheet formation is prerequisite to aggregation

Amyloid-like aggregation of alpha-synuclein and deposit in Lewy bodies are thought to be the major cause of Parkinson's disease. Here we describe the secondary structural transformation and aggregation of human alpha-synuclein and its C-terminus truncated fragments in trifluoroethanol. Proteins containing the NAC (non-amyloid component) segment undergo a three-state transition: from native random coil to beta-sheet and to alpha-helical structure, while the NAC deficient fragment and gamma-synuclein undergo a typical two-state coil-to-alpha transition. The beta-sheet form is highly hydrophobic that strongly binds to 1-anilinonaphthalene-8-sulfonic acid (ANS) and is prone to self-aggregation. The results suggest that the NAC sequence is essential to beta-sheet formation and the aggregation originates from the beta-sheet intermediate, which may be implicated in the pathogenesis of Parkinson's disease.     

Effects of oligomerization and secondary structure on the surface behavior of pulmonary surfactant proteins SP-B and SP-C

The relationship among protein oligomerization, secondary structure at the interface, and the interfacial behavior was investigated for spread layers of native pulmonary surfactant associated proteins B and C. SP-B and SP-C were isolated either from butanol or chloroform/methanol lipid extracts that were obtained from sheep lung washings. The proteins were separated from other components by gel exclusion chromatography or by high performance liquid chromatography. SDS gel electrophoresis data indicate that the SP-B samples obtained using different solvents showed different oligomerization states of the protein. The CD and FTIR spectra of SP-B isolated from all extracts were consistent with a secondary structure dominated by alpha-helix. The CD and FTIR spectra of the first SP-C corresponded to an alpha-helical secondary structure and the spectra of the second SP-C corresponded to a mixture of alpha-helical and beta-sheet conformation. In contrast, the spectra of the third SP-C corresponded to antiparallel beta-sheets. The interfacial behavior was characterized by surface pressure/area (pi-A) isotherms. Differences in the oligomerization state of SP-B as well as in the secondary structure of SP-C all produce significant differences in the surface pressure/area isotherms. The molecular cross sections determined from the pi-A isotherms and from dynamic cycling experiments were 6 nm(2)/dimer molecule for SP-B and 1.15 nm(2)/molecule for SP-C in alpha-helical conformation and 1.05 nm(2)/molecule for SP-C in beta-sheet conformation. Both the oligomer ratio of SP-B and the secondary structure of SP-C strongly influence organization and behavior of these proteins in monolayer assemblies. In addition, alpha-helix --> beta-sheet conversion of SP-C occurs simply by an increase of the summary protein/lipid concentration in solution.     

A Fourier transform infrared spectroscopic (FTIR) study of porcine and bovine pancreatic phospholipase A2 and their interaction with substrate analogues and a transition-state inhibitor

Fourier transform infrared spectroscopy has been used to investigate the secondary structure of porcine and bovine pancreatic phospholipase A2 (PLA2) and the zymogen of porcine PLA2, prophospholipase A2 (proPLA2), in both H2O and D2O media. Detailed qualitative analysis was made of these proteins using second derivative and deconvolution techniques. Quantitative studies of the proteins in solution made using Factor Analysis gave average values of 54% alpha-helix, 15% beta-sheet and 23% beta-turns. These values agree well with the secondary structures deduced from previous studies of single crystals using X-ray techniques. No significant differences in secondary structure were observed for porcine pancreatic (pro)phospholipase A2 in the presence or absence of Ca2+ ions, or in the temperature range 10-45 degrees C. The binding of the non-degradable phospholipid analogue, n-alkylphosphocholine, in monomeric form produced no significant difference in the secondary structure of either enzyme. Conformational differences were, however, observed between the enzyme lyophilised in a solid film and in aqueous solution. The change is probably due to the formation of beta-sheet upon hydration, coupled with a loss of random structures. Conformational differences in both porcine and bovine pancreatic PLA2 were observed on binding to n-alkylphosphocholine micelles. This change may be due to a small increase in the alpha-helical structure and a decrease in the beta-sheet, and/or possibly beta-turn content. Similar conformational changes were observed for the interaction of porcine and bovine PLA2 with the substrate analogue inhibitor 1-heptanoyl-2-heptanoylamino-2-deoxy-sn-glycero-3-phospho glycol in micellar form.     

Conformational behavior of ionic self-complementary peptides

Several de novo designed ionic peptides that are able to undergo conformational change under the influence of temperature and pH were studied. These peptides have two distinct surfaces with regular repeats of alternating hydrophilic and hydrophobic side chains. This permits extensive ionic and hydrophobic interactions resulting in the formation of stable beta-sheet assemblies. The other defining characteristic of this type of peptide is a cluster of negatively charged aspartic or glutamic acid residues located toward the N-terminus and positively charged arginine or lysine residues located toward the C-terminus. This arrangement of charge balances the alpha-helical dipole moment (C --> N), resulting in a strong tendency to form stable alpha-helices as well. Therefore, these peptides can form both stable alpha-helices and beta-sheets. They are also able to undergo abrupt structural transformations between these structures induced by temperature and pH changes. The amino acid sequence of these peptides permits both stable beta-sheet and alpha-helix formation, resulting in a balance between these two forms as governed by the environment. Some segments in proteins may also undergo conformational changes in response to environmental changes. Analyzing the plasticity and dynamics of this type of peptide may provide insight into amyloid formation. Since these peptides have dynamic secondary structure, they will serve to refine our general understanding of protein structure.     

Free-energy landscape of a chameleon sequence in explicit water and its inherent alpha/beta bifacial property

A sequence in yeast MATalpha2/MCM1/DNA complex that folds into alpha-helix or beta-hairpin depending on the surroundings has been known as "chameleon" sequence. We obtained the free-energy landscape of this sequence by using a generalized-ensemble method, multicanonical molecular dynamics simulation, to sample the conformational space. The system was expressed with an all-atom model in explicit water, and the initial conformation for the simulation was a random one. The free-energy landscape demonstrated that this sequence inherently has an ability to form either alpha or beta structure: The conformational distribution in the landscape consisted of two alpha-helical clusters with different packing patterns of hydrophobic residues, and four beta-hairpin clusters with different strand-strand interaction patterns. Narrow pathways connecting the clusters were found, and analysis on the pathways showed that a compact structure formed at the N-terminal root of the chameleon sequence controls the cluster-cluster transitions. The free-energy landscape indicates that a small conditional change induces alpha-beta transitions. Additional unfolding simulations done with replacing amino acids showed that the chameleon sequence has an advantage to form an alpha-helix. Current study may be useful to understand the mechanism of diseases resulting from abnormal chain folding, such as amyloid disease.     

Alanine-scanning mutagenesis of the beta-sheet region of phage T4 lysozyme suggests that tertiary context has a dominant effect on beta-sheet formation

In general, alpha-helical conformations in proteins depend in large part on the amino acid residues within the helix and their proximal interactions. For example, an alanine residue has a high propensity to adopt an alpha-helical conformation, whereas that of a glycine residue is low. The sequence preferences for beta-sheet formation are less obvious. To identify the factors that influence beta-sheet conformation, a series of scanning polyalanine mutations were made within the strands and associated turns of the beta-sheet region in T4 lysozyme. For each construct the stability of the folded protein was reduced substantially, consistent with removal of native packing interactions. However, the crystal structures showed that each of the mutants retained the beta-sheet conformation. These results suggest that the structure of the beta-sheet region of T4 lysozyme is maintained to a substantial extent by tertiary interactions with the surrounding parts of the protein. Such tertiary interactions may be important in determining the structures of beta-sheets in general.     

Solid-state NMR studies of the prion protein H1 fragment.

Conformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation-dependent chemical-shift measurements and rotational-resonance distance measurements to analyze the conformation of solid-state peptides lacking long-range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid-state NMR spectra of specifically 13C-enriched samples of H1, residues 109-122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross-polarization and magic-angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a beta-sheet conformation in the region corresponding to residues 112-121, whereas samples lyophilized from hexafluoroisopropanol display shifts indicative of alpha-helical secondary structure in the region corresponding to residues 113-117. Complete conversion to the helical conformation was not observed and conversion from alpha-helix back to beta-sheet, as inferred from the solid-state NMR spectra, occurred when samples were exposed to water. Rotational-resonance experiments were performed on seven doubly 13C-labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly beta-strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.     

gamma-Zein secondary structure in solution by circular dichroism

The proline-rich N-terminal domain of gamma-zein has been reported in relevant processes, which include its ability to cross the cell membranes. Evidences indicate that synthetic hexapeptide (PPPVHL), naturally found in N-terminal portion of gamma-zein, can adopt the polyproline II (PPII) conformation in aqueous solution. The secondary structure of gamma-zein in maize protein bodies had been analyzed by solid state Fourier transform infrared and nuclear magnetic resonance spectroscopies. However, it was not possible to measure PPII content in physiological environment since the beta-sheet and PPII signals overlap in both solid state techniques. Here, the secondary structure of gamma-zein has been analyzed by circular dichroism in SDS aqueous solution with and without ditiothreitol (DTT), and in 60% of 2-propanol and water with DTT. The results show that gamma-zein has high helical content in all solutions. The PPII conformation was present at about 7% only in water/DTT solution.     

Conformational change from antiparallel beta-sheet to alpha-helix in a series of depsipeptide, -(Leu-Leu-Lac)(n)-: syntheses, spectroscopic studies, and crystal structures of Boc-Leu-Lac-OEt and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2)

The depsipeptides Boc-Leu-Lac-OEt (1) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2) (2 and 3, respectively) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized and studied by crystallographic, CD spectroscopic, and ESI-MS analyses. In the packing cells, those three compounds adopt beta-strand conformations. Each molecule is linked into a dimer (1) or an infinite assembly (2 and 3) by tight hydrogen bonds of the type NH...O==C. Interestingly, the hexamer, 3 shows the first example of antiparallel pleated beta-sheet crystal structure for a depsipeptide molecule. In the packing cells, especially for 3, the ester groups O--C==O are perpendicularly oriented to the amide groups NH--C==O and beta-sheet planes to avoid the interaction between --O--(ester) and O==C. Therefore, when the chain length become longer, the O...O==C repulsion interaction works as a beta-sheet breaker and hence promotes an alpha-helical structure as observed for Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (4) (Oku et al. Biopolymers 2004, 75, 242-254) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 4-6) (5-7) (Katakai et al., Biopolymers 1996, 38, 285-290), in which the O...O==C repulsion does not cause significant structural changes in alpha-helical main chains. Therefore from the structural and spectroscopic analyses, we have found governing factors for the specificity in the beta-sheet and alpha-helix decision in this series of depsipeptides, -(Leu-Leu-Lac)(n)-.     

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.     

Conformational change of chaperone Hsc70 upon binding to a decapeptide: a circular dichroism study.

The conformation of bovine Hsc70, a 70-kDa heat shock cognate protein, and its conformational change upon binding to decapeptides, was studied by CD spectroscopy and secondary structure prediction (Chou, P.Y. & Fasman, G.D., 1974, Biochemistry 13, 222-245). The CD spectra were analyzed by the LINCOMB method, as well as by the convex constraint analysis (CCA) method (Perczel, A., Park, K., & Fasman, G.D., 1992, Anal. Biochem. 203, 83-93). The result of the CD analysis of Hsc70 (15% alpha-helix, 24% beta-sheet, 24% beta-turn, and 38% remainder) was very similar to the predicted secondary structure for the beta-sheet (24%) and the beta-turn (29%). However, there is disagreement between the alpha-helical content by CD analysis (15%) and the predicted structure (30%). In spite of the fact that the decapeptides contained a considerable amount of beta-sheet (22%), the interaction of the heat shock protein with the peptide resulted in an overall decrease in the content of beta-sheet conformation (-15%) of the complex. This may be due to induction of a molten globule state. The result of the CCA analysis indicated that the Hsc70 undergoes a conformational change upon binding the decapeptides.     

Stabilization of discordant helices in amyloid fibril-forming proteins

Several proteins and peptides that can convert from alpha-helical to beta-sheet conformation and form amyloid fibrils, including the amyloid beta-peptide (Abeta) and the prion protein, contain a discordant alpha-helix that is composed of residues that strongly favor beta-strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but Abeta apparently lacks such interactions. The helical propensity of the Abeta discordant region (K16LVFFAED23) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho-L-serine likewise increases the alpha-helical content of Abeta(12-28) and reduces aggregation and fibril formation of Abeta(1-40), Abeta(12-28), Abeta(12-24), and Abeta(14-23). In contrast, tripeptides with all-neutral, all-acidic or all-basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on Abeta secondary structure or fibril formation. These data suggest that in free Abeta, the discordant alpha-helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane-associated precursor protein) and that stabilization of this helix can reduce fibril formation.     

Early stages of LDL oxidation: apolipoprotein B structural changes monitored by infrared spectroscopy

Changes in the conformation of apoliprotein B-100 in the early stages of copper-mediated low density lipoprotein oxidation have been monitored by infrared spectroscopy. During the lag phase no variation in structure is observed, indicating that copper binding to the protein does not significantly affect its structure. In the propagation phase, while hydroperoxides are formed but the protein is not modified, no changes in secondary structure are observed, but the thermal profile of the band corresponding to alpha-helix is displaced in frequency, indicating changes in tertiary structure associated with this conformation but not with beta-sheet components. When aldehyde formation starts, a decrease of approximately 3% in the area of bands corresponding to alpha-helix and beta-sheet is produced, concomitantly with an increase in beta-turns and unordered structure. The two bands corresponding to beta-turns vary as well under these conditions, indicating changes in these structures. Also at this stage the thermal profile shows variations in frequency for the bands corresponding to both alpha-helix and beta-sheet.The results are consistent with the hypothesis that as soon as the polyunsaturated fatty acids from the particle core are modified, this change is reflected at the surface, in the alpha-helical components contacting the monolayer.     

Raman spectroscopic study of mechanically deformed poly-L-alanine

Raman spectroscopy has been used in investigating the conformational transitions of poly-L -alanine (PLA) induced by mechanical deformation. We see evidence of the alpha-helical, antiparallel beta-sheet, and a disordered conformation in PLA. The disordered conformation has not been discussed in previous infrared and X-ray diffraction investigations and may have local order similar to the left-handed 3₁ poly glycine helix. The amide III mode in the Raman spectrum of PLA is more sensitive than the amide I and II modes to changes in secondary structure of the polypeptide chain. Several lines below 1200 cm^?1 are conformationally sensitive and may generally be useful in the analysis of Raman spectra of proteins. A line at 909 cm^?1 decreases in intensity after deformation of PLA. In general only weak scattering is observed around 900 cm^?1 in the Raman spectra of antiparallel beta-sheet polypeptides. The Raman spectra of the amide N–H deuterated PLA and poly-L -leucine (PLL) in the alpha-helical conformation and poly-L -valine (PLV) in the beta-sheet conformation are presented. Splitting is observed in the amide III mode of PLV and the components of this mode are assigned. The Raman spectrum of an alpha-helical random copolymer of L -leucine and L -glutamic acid is shown to be consistent with the spectra of other alphahelical polypeptides.