A thermodynamic approach to the conformational preferences of the 180–195 segment derived from the human prion protein α2‐helix

On consideration that intrinsic structural weakness could affect the segment spanning the α2‐helical residues 173–195 of the PrP, we have investigated the conformational stabilities of some synthetic Ala‐scanned analogs of the peptide derived from the 180–195 C‐terminal sequence, using a novel approach whose theoretical basis originates from protein thermodynamics. Even though a quantitative comparison among peptides could not be assessed to rank them according to the effect caused by single amino acid substitution, as a general trend, all peptides invariably showed an appreciable preference for an α‐type organization, consistently with the fact that the wild‐type sequence is organized as an α‐helix in the native protein. Moreover, the substitution of whatever single amino acid in the wild‐type sequence reduced the gap between the α‐ and the β‐propensity, invariably enhancing the latter, but in any case this gap was larger than that evaluated for the full‐length α2‐helix‐derived peptide. It appears that the low β‐conformation propensity of the 180–195 region depends on the simultaneous presence of all of the Ala‐scanned residues, indirectly confirming that the N‐terminal 173–179 segment could play a major role in determining the chameleon conformational behavior of the entire 173–195 region in the PrP. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Fourier transform vibrational circular dichroism as a decisive tool for conformational studies of peptides containing tyrosyl residues

Previous UV-circular dichroism (UV-CD) and NMR studies showed that Ac-AAAAAAAEAAKA-NH(2) has an alpha-helical structure in 50% (v/v) aqueous trifluoroethanol. Replacement of Ala(1) to Ala(6) with Tyr results in spectra that show an apparent loss of helicity in the same solvent. This apparent loss of helicity could be attributed to the coupling of the tyrosyl side chain chromophore with the backbone amide. However, such electronic coupling does not affect the vibrational CD (VCD) spectra. The VCD spectra of the peptides with tyrosyl residues were identical to that of the peptide containing no Tyr, which shows the same alpha-helical structure. Because it is now clear that Tyr replacement does not change the backbone conformation of peptides, UV-CD measurements should be complemented by VCD to determine the secondary structure when electronic effects can disturb the UV-CD spectrum of the inherent structure.     

Recent contributions of electronic circular dichroism to the investigation of oligopeptide conformations

Recent applications in our laboratories of electronic circular dichroism to the study of peptide secondary structures and their changes under external stimuli are briefly reviewed. More specifically, this article deals with: 1). characterization of a novel peptide conformation; 2). origin of amino acid homo-chirality on Earth; 3). bend and helical peptides as spacers; and 4). transfer and propagation of chirality in peptides.     

Determination of alpha-helix N1 energies after addition of N1, N2, and N3 preferences to helix/coil theory

Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the alpha-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol(-1) compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol(-1) less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.     

Structures of intact glycoproteins from vibrational circular dichroism

Vibrational circular dichroism (VCD) spectra for the glycoproteins alpha1-acid glycoprotein (AGP) and bovine submaxillary mucin (BSM), have been measured in D2O solutions and for the films prepared from aqueous (H2O) buffer solutions in the 1800 to 900 cm(-1) region. The solution VCD results revealed that AGP has beta-sheet structure, along with a significant amount of alpha-helix as evidenced from a W pattern in the amide I region. The VCD of BSM solution suggested a polyproline II type structure, characterized by the appearance of strong negative couplet in the amide I region. The film VCD results on AGP and BSM suggested that the secondary structures of polypeptide fold in the film state are similar to those in the solution. The absence of any significant film VCD in the low frequency region (1200-900 cm(-1)), suggested that the dominant linkage for carbohydrate residues is likely to be a beta linkage. VCD spectroscopy gains importance in the secondary structural analysis of polypeptide fold in glycoproteins due to the absence of interfering VCD from the carbohydrate residues in the conformationally sensitive amide I region. Also, film VCD studies permit measurements in the low wavenumber region (1200-900 cm(-1)) that reveal the dominant type of linkage for carbohydrate residues. Such clear structural information is unlike that from ECD, where ECD bands of acylated amino sugar residues interfere with those of polypeptide backbone in the conformationally sensitive far-UV region.     

Exploring the protein G helix free-energy surface by solute tempering metadynamics

The free-energy landscape of the alpha-helix of protein G is studied by means of metadynamics coupled with a solute tempering algorithm. Metadynamics allows to overcome large energy barriers, whereas solute tempering improves the sampling with an affordable computational effort. From the sampled free-energy surface we are able to reproduce a number of experimental observations, such as the fact that the lowest minimum corresponds to a globular conformation displaying some degree of beta-structure, that the helical state is metastable and involves only 65% of the chain. The calculations also show that the system populates consistently a pi-helix state and that the hydrophobic staple motif is present only in the free-energy minimum associated with the helices, and contributes to their stabilization. The use of metadynamics coupled with solute tempering results then particularly suitable to provide the thermodynamics of a short peptide, and its computational efficiency is promising to deal with larger proteins.     

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.     

Protein recognition motifs: design of peptidomimetics of helix surfaces

Helices represent one of the most common recognition motifs in proteins. The design of nonpeptidic scaffolds, such as the 3,2',2'-tris-substituted terphenyl, that can imitate the side-chain orientation along one face of an alpha-helix potentially provides an effective means to modulate helix-recognition functions. Here, based on theoretical arguments, we described novel alpha-helix mimetics which are more effective than the terphenyl at constraining the aryl-aryl torsion angles to those associated with structures suitable for mimicking the alpha-helical twist for side-chain orientation and for superimposing the side chains of residues i, i + 3 or i + 4, i + 7 when compared with the alpha-beta side-chain vectors of the regular alpha-helix with an improved root mean square deviation (RMSD) of approximately 0.5 A. In addition, this study suggests that rotamer distributions around the C(alpha)--C(beta) bonds of these helix mimetics are similar to those of alpha-helices, except that these rotamer distributions show an approximately 60 degrees shift compared to those of alpha-helices when the mimetic axis is superimposed upon the helix axis. This change in rotamer orientation complicates mimicry of the helix surface.     

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.     

On the helix sense of gramicidin A single channels.

In order to resolve whether gramicidin A channels are formed by right- or left-handed beta-helices, we synthesized an optically reversed (or mirror image) analogue of gramicidin A, called gramicidin A-, to test whether it forms channels that have the same handedness as channels formed by gramicidin M- (F. Heitz et al., Biophys. J. 40:87-89, 1982). In gramicidin M- the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therefore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of gramicidin M- in dimyristoylphosphatidylcholine vesicles is consistent with a left-handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149-160, 1986), and the CD spectra of gramicidins A and A- are essentially mirror images of each other. Based on hybrid channel experiments, gramicidin A- and M- channels are structurally equivalent, while gramicidin A and A- channels are nonequivalent, being of opposite helix sense. Gramicidin A- channels are therefore left-handed, and natural gramicidin A channels in phospholipid bilayers are right-handed beta 6.3-helical dimers.     

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.     

Disruption of the beta-sheet structure of a protected pentapeptide, related to the beta-amyloid sequence 17-21, induced by a single, helicogenic C(alpha)-tetrasubstituted alpha-amino acid.

Fifteen years ago it was shown that an alpha-aminoisobutyric acid (Aib) residue is significantly more effective than an L-Pro or a D-amino acid residue in inducing beta-sheet disruption in short model peptides. As this secondary structure element is known to play a crucial role in the neuropathology of Alzheimer's disease, it was decided to check the effect of Aib (and other selected, helix inducer, C(alpha)-tetrasubstituted alpha-amino acids) on the beta-sheet conformation adopted by a protected pentapeptide related to the sequence 17-21 of the beta-amyloid peptide. By use of FT-IR absorption and 1H NMR techniques it was found that the strong self-association characterizing the pentapeptide molecules in weakly polar organic solvents is completely abolished by replacing a single residue with Aib or one of its congeners.     

Determination of protein fold class from Raman or Raman optical activity spectra using random forests

Knowledge of the fold class of a protein is valuable because fold class gives an indication of protein function and evolution. Fold class can be accurately determined from a crystal structure or NMR structure, though these methods are expensive, time-consuming, and inapplicable to all proteins. In contrast, vibrational spectra [infra-red, Raman, or Raman optical activity (ROA)] are rapidly obtained for proteins under wide range of biological molecules under diverse experimental and physiological conditions. Here, we show that the fold class of a protein can be determined from Raman or ROA spectra by converting a spectrum into data of 10 cm⁻¹ bin widths and applying the random forest machine learning algorithm. Spectral data from 605 and 1785 cm⁻¹ were analyzed, as well as the amide I, II, and III regions in isolation and in combination. ROA amide II and III data gave the best performance, with 33 of 44 proteins assigned to one of the correct four top-level structural classification of proteins (SCOP) fold class (all α, all β, α and β, and disordered). The method also shows which spectral regions are most valuable in assigning fold class.     

C(alpha)-hydroxymethyl methionine: synthesis, optical resolution and crystal structure of its (+)-N(alpha)-benzoyl derivative.

(R, S)-Methionine was transformed into C(alpha)-hydroxymethyl methionine by a route involving C(alpha)-hydroxymethylation of 2-phenyl-4-methylthioethyl-5-oxo-4,5-dihydro-1,3-oxazole. The absolute configuration of (-)-C(alpha)-hydroxymethyl methionine was elucidated to be (S) by chemical correlation with (S) (-)-C(alpha)-ethyl serine. Absolute structure determination (by single crystal X-ray diffraction) on N(alpha)-benzoyl-C(alpha)-hydroxymethyl methionine confirmed the (R)-configuration for the (+)-enantiomer. In addition, the X-ray diffraction analysis showed that the C(alpha,alpha)-disubstituted glycyl residue adopts the fully extended (C5) conformation.     

The prion protein unstructured N-terminal region is a broad-spectrum molecular sensor with diverse and contrasting potential functions

The physiological function of the prion protein (PrP(C) ) and its conversion into its infectious form (PrP(Sc) ) are central issues to understanding the pathogenesis of prion diseases. The N-terminal moiety of PrP(C) (NH(2) -PrP(C) ) is an unstructured region with the characteristic of interacting with a broad range of partners. These interactions endow PrP(C) with multifunctional and sometimes contrasting capabilities, including neuroprotection and neurotoxicity. Recently, binding of β-sheet rich conformers to NH(2) -PrP(C) demonstrated a probable neurotoxic function for PrP(C) in Alzheimer's disease. NH(2) -PrP(C) also enhances the propagation of prions in vivo and is the target of the most potent antiprion compounds. Another level of complexity is provided by endoproteolysis and release of most of NH(2) -PrP(C) into the extracellular space. Further studies will be necessary to understand how NH(2) -PrP(C) regulates the physiological function of PrP(C) and how it is involved in the corruption of its normal function in diseases.     

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2alpha on CK2beta with an upshift of the CK2alpha melting temperature of more than 9 degrees . Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2alpha and CK2beta is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2beta construct to 2.8 A resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2beta conformation is largely conserved upon association with CK2alpha, whereas the latter undergoes significant structural adaptations of its backbone.     

A synthetic analog of plantaricin 149 inhibiting food-borne pathogenic bacteria:evidence for alpha-helical conformation involved in bacteria-membrane interaction.

Plantaricin-149 is a bacteriocin produced by Lactobacillus plantarum NRIC 149 (a LAB isolated from pineapple), which consists of a peptidic chain made up of 22 amino acid residues [Kato et al. J. Ferment. Bioeng. 1994; 77: 277-282]. In this work, a synthetic C-terminal amidated peptide analog denoted Pln149a was prepared by SPPS-Fmoc chemistry and the antagonistic activity against gram-positive and gram-negative bacteria was tested. The secondary structure was studied by circular dichroism (CD) and the vicinity of the tyrosine residue by fluorescence spectroscopy under different conditions. We report the results of the interaction of Pln149a with reverse micelles prepared from the amphiphilic AOT in cyclohexane.Synthetic plantaricin was active against one strain of Staphylococcus aureus and four strains of Listeria genus at pH 5.5 and 7.4 and, like its natural variant, inhibited L. plantarum ATCC 8014.The data derived from spectroscopic measurements in presence of AOT reverse micelles suggest that the secondary structure of the peptide upon interaction is an alpha-helix. In this membrane model, the hydrophobic side of the alpha-helix is inserted into the micelles, leaving the lysines exposed to the solvent and interacting with the polar moieties of AOT. The fluorescence data point out that the N-terminal tyrosine residue is close to the micellar interface.     

Cooperative effects in hydrogen-bonding of protein secondary structure elements: a systematic analysis of crystal data using Secbase

A systematic analysis of the hydrogen-bonding geometry in helices and beta sheets has been performed. The distances and angles between the backbone carbonyl O and amide N atoms were correlated considering more than 1500 protein chains in crystal structures determined to a resolution better than 1.5 A. They reveal statistically significant trends in the H-bond geometry across the different secondary structural elements. The analysis has been performed using Secbase, a modular extension of Relibase (Receptor Ligand Database) which integrates information about secondary structural elements assigned to individual protein structures with the various search facilities implemented into Relibase. A comparison of the mean hydrogen-bond distances in alpha helices and 3(10) helices of increasing length shows opposing trends. Whereas in alpha helices the mean H-bond distance shrinks with increasing helix length and turn number, the corresponding mean dimension in 3(10) helices expands in a comparable series. Comparing similarly the hydrogen-bond lengths in beta sheets there is no difference to be found between the mean H-bond length in antiparallel and parallel beta sheets along the strand direction. In contrast, an interesting systematic trend appears to be given for the hydrogen bonds perpendicular to the strands bridging across an extended sheet. With increasing number of accumulated strands, which results in a growing number of back-to-back piling hydrogen bonds across the strands, a slight decrease of the mean H-bond distance is apparent in parallel beta sheets whereas such trends are obviously not given in antiparallel beta sheets. This observation suggests that cooperative effects mutually polarizing spatially well-aligned hydrogen bonds are present either in alpha helices and parallel beta sheets whereas such influences seem to be lacking in 3(10) helices and antiparallel beta sheets.