Characterization of the denatured structure of pyrrolidone carboxyl peptidase from a hyperthermophile under nondenaturing conditions: role of the C-terminal alpha-helix of the protein in folding and stability

The cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile, Pyrococcus furiosus, can be trapped in the denatured state under nondenaturing conditions, corresponding to the denatured structure that exists in equilibrium with the native state under physiological conditions. The denatured state is the initial state (D1 state) in the refolding process but differs from the completely denatured state (D2 state) in the concentrated denaturant. Also, it has been found that the D1 state corresponds to the heat-denatured state. To elucidate the structural basis of the D1 state, H/D exchange experiments with PCP-0SH were performed at pD 3.4 and 4 degrees C. The results indicated that amide protons in the C-terminal alpha6-helix region hardly exchanged in the D1 state with deuterium even after 7 days, suggesting that the alpha6-helix (from Ser188 to Glu205) of PCP-0SH was stably formed in the D1 state. In order to examine the role of the alpha6-helix in folding and stability, H/D exchange experiments with a mutant, A199P, at position 199 in the alpha6-helix region were performed. The alpha6-helix region of A199P in the D1 state was partially unprotected, while some hydrophobic residues were protected against the H/D exchange, although these hydrophobic residues were unprotected in the wild-type protein. These results suggest that the structure of A199P in the D1 state formed a temporary stable denatured structure with a non-native hydrophobic cluster and the unstructured alpha6-helix. Both the stability and the refolding rate decreased by the substitution of Pro for Ala199. We can conclude that the native-like helix (alpha6-helix) of PCP-0SH is already constructed in the D1 state and is necessary for efficient refolding into the native structure and stabilization of PCP-0SH.     

Conformational properties of five peptides corresponding to the entire sequence of glutathione transferase domain II

Five peptides matching the helices alpha4, alpha5, alpha6, alpha7, and alpha8, spanning the entire sequence of domain II of pG-STP1-1, have been synthesized and their conformations analyzed by far-UV CD spectroscopy. The results show that a5, a7, and a8 peptides are unstructured in water/2,2,2-trifluoroethanol (TFE) solutions. The a4-peptide also adopts random conformations in aqueous solvent. Moreover, the relative low helical content (20%), estimated for this peptide in the presence of 30% (v/v) TFE, suggests that the sequence of this protein fragment does not possess sufficient information for a strong helical propensity. On the contrary, the synthesized a6 peptide, in the presence of TFE, showed a relevant structural autonomy with a helical content (41%) which was significantly higher than that estimated, under the same conditions, for all other peptides. More in general in the presence of solvents less polar than water, the isolated a6 peptide shows the same helical conformation adopted by the corresponding alpha6-helix in the hydrophobic core of the protein. A n-capping box motif, strictly conserved at the N-terminal of the alpha6-helix of all GST and related protein including eucaryotic translation elongation factor (EF1gamma) and the yeast prion protein Ure2, plays an important role in the alpha-helix nucleation and stability of this protein fragment. The results suggest that the alpha6-helix might represent a nucleation site of GST folding and that the helical conformation of this region of the protein is an important requirement during earlier events of GST refolding.     

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.     

Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies

The refolding rate of heat-denatured cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus has been reported to be unusually slow under some conditions. To elucidate the structural basis of the unusually slow kinetics of the protein, the denaturation and refolding processes of the PCP-0SH were investigated using a real-time 2D (1)H-(15)N HSQC and CD experiments. At 2 M urea denaturation of the PCP-0SH in the acidic region, all of the native peaks in the 2D HSQC spectrum completely disappeared. The conformation of the PCP-0SH just after removal of 6 M GuHCl could be observed as a stable intermediate (D(1) state) in 2D HSQC and CD experiments, which is similar to a molten globule structure. The D(1) state of the PCP-0SH, which is the initial state of refolding, corresponded to the state at 2 M urea and seemed to be the denatured state in equilibrium with the native state under the physiological conditions. The refolding of PCP-0SH from the D(1) state to the native state could be observed to be highly cooperative without any intermediates between them, even if the refolding rate was quite slow. In the higher concentration of denaturants, PCP-0SH showed HSQC and CD spectra characteristic of completely unfolded proteins called the D(2) state. The unusually slow refolding rate was discussed as originating in the conformations in the transition state and/or the retardation of reorganization in an ensemble of nonrandom denatured structures in the D(1) state.     

Structural characterization of a trapped folding intermediate of pyrrolidone carboxyl peptidase from a hyperthermophile

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix-helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central β-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH.     

Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T1.

Trifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the alpha-helix of ribonuclease T1. There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T1 helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine-based peptides and the ribonuclease T1 peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context.     

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.     

Helix propagation and N-cap propensities of the amino acids measured in alanine-based peptides in 40 volume percent trifluoroethanol.

The helix propagation and N-cap propensities of the amino acids have been measured in alanine-based peptides in 40 volume percent trifluoroethanol (40% TFE) to determine if this helix-stabilizing solvent uniformly affects all amino acids. The propensities in 40% TFE are compared with revised values of the helix parameters of alanine-based peptides in water. Revision of the propensities in water is the result of redefining the capping statistical weights and evaluating the helix nucleation constant with N-capping explicitly included in the helix-coil model. The propagation propensities of all amino acids increase in 40% TFE relative to water, but the increases are highly variable. In water, all beta-branched and beta-substituted amino acids are helix breakers. In 40% TFE, the propagation propensities of the nonpolar amino acids increase greatly, leaving charged and neutral polar, beta-substituted amino acids as helix breakers. Glycine and proline are strong helix breakers in both solvents. Free energy differences for helix propagation (delta delta G) between alanine and other nonpolar amino acids are twice as large in water as predicted from side-chain conformational entropies, but delta delta G values in 40% TFE are close to those predicted from side-chain entropies. This dependence of delta delta G on the solvent points to a specific role of water in determining the relative helix propensities of the nonpolar amino acids. The N-cap propensities converge toward a common value in 40% TFE, suggesting that differential solvation by water contributes to the diversity of N-cap values shown by the amino acids.     

Thermodynamics of binding by calmodulin correlates with target peptide α‐helical propensity

In this work, we have examined contributions to the thermodynamics of calmodulin (CaM) binding from the intrinsic propensity for target peptides to adopt an α‐helical conformation. CaM target sequences are thought to commonly reside in disordered regions within proteins. Using the ability of TFE to induce α‐helical structure as a proxy, the six peptides studied range from having almost no propensity to adopt α‐helical structure through to a very high propensity. This despite all six peptides having similar CaM‐binding affinities. Our data indicate there is some correlation between the deduced propensities and the thermodynamics of CaM binding. This finding implies that molecular recognition features, such as CaM target sequences, may possess a broad range of propensities to adopt local structure. Given that these peptides bind to CaM with similar affinities, the data suggest that having a higher propensity to adopt α‐helical structure does not necessarily result in tighter binding, and that the mechanism of CaM binding is very dependent on the nature of the substrate sequence. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Characterization of amyloidogenic intermediate states through a combined use of CD and NMR spectroscopy

Characterization of amyloidogenic intermediate states is of central importance in understanding the molecular mechanism of amyloid formation. In this study, we utilized CD and NMR spectroscopy to investigate secondary structure of the monomeric amyloidogenic intermediate of a β-structured SH3 domain, which was induced by trifluoroethanol (TFE). The combined biophysical studies showed that the native state SH3 domain is gradually converted to the amyloidogenic intermediate state at TFE concentrations of 20-26% (v/v) and the aggregation-prone state contains substantial amount of the β-sheet conformation (∼ 30%) with disordered (54%) and some helical characters (16%). Under weaker amyloidogenic conditions of higher TFE concentrations (> 40%), the β-sheet structures were gradually changed to helical conformations and the relative content of the helical and β-sheet conformations was highly correlated with the aggregation propensity of the SH3 domain. This indicates that the β-sheet characters of the amyloidogenic states may be critical to the effective amyloid formation.     

Conformational studies of Aib-rich peptides containing lactam-bridged side chains: evidence of 3(10)-helix formation

Aib-rich side-chain lactam-bridged oligomers Ac-(Glu-Aib-Aib-Lys)n-Ala-OH with n = 1,2,3 were designed and synthesized as putative models of the 3(10)-helix. The lactam bridge between the side chains of L-Glu and L-Lys in (i)--(i + 3) positions was introduced in order to enhance the structural preference toward the right-handed 3(10)-helix. The conformational properties of the three peptides were studied in trifluoroethanol (TFE) solution by CD, NMR, and computer simulations. The structural information was derived mainly from the analysis of nuclear Overhauser effect spectroscopy spectra. The presence of alpha H(i)-HN(i + 2) and of alpha H(i)-HN(i + 3) connectivities and the absence of alpha H(i)-HN(i + 4) connectivities indicate that these peptides fold into a 3(10)-helix rather than into an alpha-helix. Based on these conformational features, stereospecific assignment of the Aib methyl groups was possible. The results of such experiments and of the subsequent distance geometry and restrained molecular dynamics simulations reveal a marked preference of these peptides for 3(10)-helix. The CD spectra of these peptides indicate that the helix content increases upon chain elongation. The CD spectrum of the trimer is characterized by a negative band at 200 nm and by a weak positive band around 220 nm. The CD spectrum in TFE is different from that observed in aqueous solution in the presence of SDS micelles, reported in our previous work, and from those reported by a different research group for 3(10)-helical peptides. A possible reason for these differences could rest in the presence of different equilibria of the conformer populations of the various peptides in different solvent systems.     

Facile transition between 3(10)- and alpha-helix: structures of 8-, 9-, and 10-residue peptides containing the -(Leu-Aib-Ala)2-Phe-Aib- fragment.

A structural transition from a 3(10)-helix to an alpha-helix has been characterized at high resolution for an octapeptide segment located in 3 different sequences. Three synthetic peptides, decapeptide (A) Boc-Aib-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, nonapeptide (B) Boc-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, and octapeptide (C) Boc-(Leu-Aib-Ala)2-Phe-Aib-OMe, are completely helical in their respective crystals. At 0.9 A resolution, R factors for A, B, and C are 8.3%, 5.4%, and 7.3%, respectively. The octapeptide and nonapeptide form ideal 3(10)-helices with average torsional angles phi(N-C alpha) and psi(C alpha-C') of -57 degrees, -26 degrees C and -60 degrees, -27 degrees for B. The 10-residue peptide (A) begins as a 3(10)-helix and abruptly changes to an alpha-helix at carbonyl O(3), which is the acceptor for both a 4-->1 hydrogen bond with N(6)H and a 5-->1 hydrogen with N(7)H, even though the last 8 residues have the same sequence in all 3 peptides. The average phi, psi angles in the decapeptide are -58 degrees, -28 degrees for residues 1-3 and -63 degrees, -41 degrees for residues 4-10. The packing of helices in the crystals does not provide any obvious reason for the transition in helix type. Fourier transform infrared studies in the solid state also provide evidence for a 3(10)- to alpha-helix transition with the amide I band appearing at 1,656-1,657 cm-1 in the 9- and 10-residue peptides, whereas in shorter sequences the band is observed at 1,667 cm-1.     

An inducible helix–Gly–Gly–helix motif in the N-terminal domain of histone H1e: A CD and NMR study

Knowledge of the structural properties of linker histones is important to the understanding of their role in higher-order chromatin structure and gene regulation. Here we study the conformational properties of the peptide Ac-EKTPVKKKARKAAGGAKRKTSG-NH(2) (NE-1) by circular dichroism and (1)H-NMR. This peptide corresponds to the positively charged region of the N-terminal domain, adjacent to the globular domain, of mouse histone H1e (residues 15-36). This is the most abundant H1 subtype in many kinds of mammalian somatic cells. NE-1 is mainly unstructured in aqueous solution, but in the presence of the secondary-structure stabilizer trifluoroethanol (TFE) it acquires an alpha-helical structure. In 90% TFE solution the alpha-helical population is approximately 40%. In these conditions, NE-1 is structured in two alpha-helices that comprise almost all the peptide, namely, from Thr17 to Ala27 and from Gly29 to Thr34. Both helical regions are highly amphipathic, with the basic residues on one face of the helix and the apolar ones on the other. The two helical elements are separated by a Gly-Gly motif. Gly-Gly motifs at equivalent positions are found in many vertebrate H1 subtypes. Structure calculations show that the Gly-Gly motif behaves as a flexible linker between the helical regions. The wide range of relative orientations of the helical axes allowed by the Gly-Gly motif may facilitate the tracking of the phosphate backbone by the helical elements or the simultaneous binding of two nonconsecutive DNA segments in chromatin.     

Amphipathic control of the 3(10)-/alpha-helix equilibrium in synthetic peptides.

A series of short, amphipathic peptides incorporating 80% C(alpha),C(alpha)-disubstituted glycines has been prepared to investigate amphipathicity as a helix-stabilizing effect. The peptides were designed to adopt 3(10)- or alpha-helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mM SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3(10)-helix in peptides containing amino acids with large side-chains, as with 1-aminocyclohexane-1-carboxylic acid (Ac(6)c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3(10)-helix.     

Completely buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles

Pyrrolidone carboxyl peptidases (PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH (C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar (E192A/I/V) and polar (E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pKa value > or = 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E --> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E --> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that (1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and (2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.     

Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway.

Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.     

Solution structure of the first and second transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the first and the second transmembrane segment of the bovine mitochondrial oxoglutarate carrier (OGC) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. Peptides 21-46 and 78-108 of its primary sequence were synthesized and structurally characterized in membrane-mimetic environments. CD data showed that at high concentrations of TFE (>50%) and SDS (>2%) both peptides assume alpha-helical structures, whereas in more hydrophilic environments only peptide 78-108 has a helical structure. (1)H-NMR spectra of the two peptides in TFE/water and SDS were fully assigned, and the secondary structures of the peptides were obtained from nuclear Overhauser effects, (3)J(alphaH-NH) coupling constants and alphaH chemical shifts. The three-dimensional solution structures of the peptides in TFE/water were generated by distance geometry calculations. A well-defined alpha-helix was found in the region K24-V39 of peptide 21-46 and in the region A86-F106 of peptide 78-108. We cannot exclude that in intact OGC the extension of these helices is longer. The helix of peptide 21-46 is essentially hydrophobic, whereas that of peptide 78-108 is predominantly hydrophilic.     

A helix-turn motif in the C-terminal domain of histone H1

The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs.     

Role of α-helical conformation of histatin-5 in candidacidal activity examined by proline variants

Human salivary histatin-5 (Hsn-5) is a potent in vitro anticandidal agent. The aim of this study was to investigate the importance of α-helical structure of Hsn-5 for its candidacidal activity. The following three Hsn-5 variants, where one or more functionally nonessential residues were replaced with proline (potent α-helix breaker), were produced by Escherichia coli expression system: H21P (1P), H19P/H21P (2P), and E16P/H19P/H21P (3P). The activities of purified proteins were determined by candidacidal assays, and the secondary structures by circular dichroism (CD) spectroscopy in trifluoroethanol (TFE) that is considered the helix-promoting solvent, and lysophosphatidyl-glycerol (LPG) micelles, the environment that more closely resembles the biological membranes. Our results indicated that 3P variant displayed a candidacidal activity which was similar to that of unaltered Hsn-5 (0P), while 1P and 2P variants showed lower cidal activity. The CD spectra in TFE indicated that 3P variant has less helical characteristics than the 0P, 1P and 2P. These results suggested that the α-helical content of Hsn-5 proline variants does not correlate with the candidacidal activity. Further, the CD spectral analysis of peptides in LPG micelles indicated the formation of β-turn structures in 0P and 3P variants. In conclusion, 3P variant which exhibited comparable candidacidal activity to 0P contains lower percentage of α-helical structure than 1P and 2P variants, which exhibited lower candidacidal activity. This suggests α-helix may not be important for anticandidal activity of Hsn-5.     

Helix propagation in trifluoroethanol solutions.

Helix propagation of the S-peptide sequence (residues 1-19 of ribonuclease A) in 2,2,2-trifluoroethanol (TFE) solutions has been investigated with CD and nmr Overhauser effect spectroscopies. In this study, the S-peptide helix is covalently initiated at the N-terminus through disulfide bonds to a helix scaffold derived from the N-terminal sequence of the bee venom peptide apamin. The entire S-peptide sequence of this hybrid sequence peptide becomes helical at high proportions of TFE. Residues 14-19 of the S-peptide are not helical in the free peptide in TFE, nor are they helical in ribonuclease A. The "helix stop" signal encoded by the S-peptide sequence near residue 13 does not persist at high TFE with this hybrid sequence peptide. The helix-stabilizing effects of TFE are due at least in part to facilitated propagation of an extant helix. This stabilizing effect appears to be a general solvation effect and not due to specific interaction of the helical peptide with TFE. Specifically these data support the idea that TFE destabilizes the coil state by less effective hydrogen bonding of the peptide amide to the solvent.