Stabilization of the ribonuclease S-peptide alpha-helix by trifluoroethanol

The effects of trifluoroethanol (TFE) on the stability of the alpha-helix formed by ribonuclease S-peptide, residues 1-19 of ribonuclease A, were studied by measuring circular dichroism as a function of TFE concentration, pH, and temperature. The S-peptide forms an unusually stable alpha-helix, which is known to be stabilized by TFE. The magnitude of the effect of charged groups on the peptide, manifested by the change in alpha-helix stability as a function of pH, was not altered significantly by either TFE concentration or temperature, indicating that the lower dielectric constant of TFE is not important in the stabilization of this alpha-helix. This suggests that the alpha-helix might be stabilized by many interactions in addition to the effects of charges. The titration curve of circular dichroism vs. TFE concentration appears to be cooperative at 0 degree C, but becomes progressively less cooperative at temperatures between 25 and 75 degrees C. The properties of the TFE stabilization indicate that TFE might be a useful probe with which to measure the stability of marginally stable peptides and small proteins.     

C-terminal analogues of parathyroid hormone: effect of C-terminus function on helical structure, stability, and bioactivity

We have studied the effects of C-terminal group modifications (amide, methylamide, dimethylamide, aldehyde, and alcohol) on the conformation, adenylyl cyclase stimulation (AC), or binding of parathyroid hormone (hPTH) analogues, hPTH(1-28)NH(2) and hPTH(1-31)NH(2). hPTH(1-31)NH(2) has a C-terminal alpha-helix bounded by residues 17-29 [Chen, Z., et al. (2000) Biochemistry 39, 12766]. In both cases, relative to the natural analogue with a carboxyl C-terminus, the amide and methylamide had increased helix content whereas the dimethylamide forms had CD spectra more similar to the carboxyl one. Conformational effects were more pronounced with hPTH(1-28) than with hPTH(1-31), with increases in helix content of approximately 30% in contrast to 10%. Stabilization of the C-terminal helix of residues 1-28 seemed to correlate with an ability of the C-terminal function to H-bond appropriately. None of the analogues affected the AC stimulating activity significantly, but there was an up to 15-fold decrease in the level of apparent binding of the carboxyl hPTH(1-28) analogue compared to that of the methylamide and a 4-fold decrease in the level of binding of the aldehyde or dimethylamide. There was no significant change in binding activities for the 1-31 analogues. These observations are consistent with previous studies that imply the importance of a region of the hormone's C-terminal alpha-helix for tight binding to the receptor. They also show that modulation of helix stability does have an effect on the binding of the hormone, but only when the C-terminus is at the putative end of the helix. The similarity of AC stimulation even when binding changed 10-fold can be explained by assuming greater efficacy of the weaker binding PTH-receptor complexes in stimulating AC.     

Role of recurrent hydrophobic residues in catalysis of helix formation by T cell-presented peptides in the presence of lipid vesicles

We tested the hypothesis that the recurrence of hydrophobic amino acids in a polypeptide at positions falling in an axial, hydrophobic strip if the sequence were coiled as an alpha helix, can lead to helical nucleation on a hydrophobic surface. The hydrophobic surface could anchor such residues, whereas the peptide sequence grows in a helical configuration that is stabilized by hydrogen bonds among carbonyl and amido NH groups along the peptidyl backbone of the helix, and by other intercycle interactions among amino acid side chains. Such bound, helical structures might protect peptides from proteases and/or facilitate transport to a MHC-containing compartment and thus be reflected in the selection of T cell-presented segments. Helical structure in a series of HPLC-purified peptides was estimated from circular dichroism measurements in: 1) 0.01 M phosphate buffer, pH 7.0, 2) that buffer with 45% trifluoroethanol (TFE), and 3) that buffer with di-O-hexadecyl phosphatidylcholine vesicles. By decreasing the dielectric constant of the buffer, TFE enhances intrapeptide interactions generally, whereas the lipid vesicles only provide a surface for hydrophobic interactions. The peptides varied in their strip-of-helix hydrophobicity indices (SOHHI; the mean Kyte-Doolittle hydrophobicities of residues in an axial strip of an alpha helix) and in proline content. Structural order for peptides with helical circular dichroism spectra was estimated as percentage helicity from circular dichroism theta 222 nm values and peptide concentration. A prototypic alpha helical peptide with three cycles plus two amino acids and an axial hydrophobic strip of four leucyl residues (SOHHI = 3.8) was disordered in phosphate buffer, 58% helical in that buffer with 48% TFE, and 36% helical in that buffer with vesicles. Percentage helicity in the presence of vesicles of the subset of peptides without proline followed their SOHHI values. Peptides with multiple prolyl residues had circular dichroism spectra with strong signals, but since they did not have altered spectra in the presence of vesicles relative to phosphate buffer alone, the hydrophobic surface of the vesicle did not appear to stabilize those structures.     

Biological properties of two models of calcitonin gene related peptide with idealized amphiphilic alpha-helices of different lengths

Previous studies on calcitonin gene related peptide (CGRP) have demonstrated that it has the characteristics of an amphiphilic peptide, and from an examination of the sequence, we have proposed that it contains an amphiphilic alpha-helix. We have synthesized two analogues of CGRP which have different lengths of idealized amphiphilic alpha-helical secondary structure. The first model, CGRM-1, has been substituted with residues generating an idealized amphiphilic alpha-helix in the region between residues 8 and 25, equivalent to approximately five turns of an alpha-helix. This peptide is not an agonist in any of our bioassays, but it does bind with low affinity to rCGRP receptors in crude liver membranes. Our second model, CGRM-2, has an idealized amphiphilic alpha-helix between residues 8 and 18, which is equivalent to approximately three turns of an alpha-helix. In an in vitro rat vas deferens assay, this peptide is an agonist with a potency one-fourth that of the native hormone. However, the potency of CGRM-2 in an adenylate cyclase assay is much lower, only 1/140th the potency of CGRP. Both model peptides display amphiphilic characteristics commensurate with their design. We conclude that there is an amphiphilic alpha-helix in rCGRP between residues 8 and 18 and that this helix terminates in the vicinity of residue 18.     

Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide sakacin P and a sakacin P variant that is structurally stabilized by an inserted C-terminal disulfide bridge

The three-dimensional structures in dodecylphosphocholine (DPC) micelles and in trifluoroethanol (TFE) of the pediocin-like antimicrobial peptide sakacin P and an engineered variant of sakacin P (termed sakP[N24C+44C]) have been determined by use of nuclear magnetic resonance spectroscopy. SakP[N24C+44C] has an inserted non-native activity- and structure-stabilizing C-terminal disulfide bridge that ties the C-terminus to the middle part of the peptide. In the presence of DPC, the cationic N-terminal region (residues 1-17) of both peptides has an S-shaped conformation that is reminiscent of a three-stranded antiparallel beta-sheet and that is more pronounced when the peptide was dissolved in TFE instead of DPC. The four positively charged residues located in the N-terminal part are found pointing to the same direction. For both peptides, the N-terminal region is followed by a well-defined central amphiphilic alpha-helix (residues 18-33), and this in turn is followed by the C-terminal tail (residues 34-43 for sakacin P and 34-44 for sakP[N24C+44C]) that lacks any apparent common secondary structural motif. In the presence of DPC, the C-terminal tails in both peptides fold back onto the central alpha-helix, thereby creating a hairpin-like structure in the C-terminal halves. The lack of long-range NOEs between the beta-sheet Nu-terminal region and the hairpin-like C-terminal half indicates that there is a flexible hinge between these regions. We discuss which implications such a structural arrangement has on the interaction with the target cell membrane.     

Conformational analysis of a mitochondrial presequence derived from the F1-ATPase beta-subunit by CD and NMR spectroscopy.

Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood. We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the presequence for the beta-subunit of F1-ATPase, a mitochondrial matrix protein. Although this peptide is essentially unstructured in water, alpha-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE). In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and alpha-helical conformations, with a significant population of alpha-helix throughout the entire peptide. The helix is somewhat more stable in the N-terminal part of the presequence (residues 4-10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV. Addition of increasing amounts of TFE causes the alpha-helical content to increase even further, and the TFE titration data for the presequence peptide of the F1-ATPase beta-subunit are not consistent with a single, cooperative transition from random coil to alpha-helix. There is evidence that helix formation is initiated in two different regions of the peptide. This result helps to explain the redundancy of the targeting information contained in the presequence for the F1-ATPase beta-subunit.     

Spectroscopic studies of a phosphoinositide-binding peptide from gelsolin: behavior in solutions of mixed solvent and anionic micelles.

The peptide G(150-169) corresponds to a phosphatidylinositol 4,5-bisphosphate (PIP2) and filamentous actin (F-actin) binding site on gelsolin (residues 150-169, with the sequence KHVVPNEVVVQRLFQVKGRR). The conformation of this peptide in trifluoroethanol (TFE) aqueous solution was determined by 1H nuclear magnetic resonance as the first step toward understanding the structural aspects of the interaction of G(150-169) and PIP2. The circular dichroism experiments show that G(150-169) adopts a predominantly alpha-helical form in both 50% TFE aqueous solution and in the presence of PIP2 micelles, therefore establishing a connection between the two conformations. 1H nuclear magnetic resonance experiments of G(150-169) in TFE co-solvent show that the helical region extends from Pro-154 to Lys-166. The amphiphilic nature of this helical structure may be the key to understanding the binding of the peptide to lipids. Sodium dodecyl sulfate micelle solution is used as a model for anionic lipid environments. Preliminary studies of the conformation of G(150-169) in sodium dodecyl sulfate micelle solution show that the peptide forms an alpha-helix similar to but with some structural differences from that in TFE co-solvent. Fluorescence experiments provide evidence of peptide clustering over a narrow range of peptide/PIP2 ratios, which is potentially relevant to the biological function of PIP2.     

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.     

Solution structure of salmon calcitonin

Salmon calcitonin, a 32-residue peptide with a 1-7 disulfide bridge, was synthesized by standard solid-phase techniques, and studied by CD and two-dimensional NMR experiments. The peptide was dissolved in pure trifluoroethanol (TFE) and in aqueous solutions containing various amounts of TFE. CD studies in pure TFE indicated the presence of an alpha-helical structure comprising 40% of the constituent amino acids. This was fully confirmed by nmr. A detailed analysis was performed with the peptide in a 9 : 1 deuterated TFE/H2O mixture. A total of 365 nuclear Overhauser enhancements (154 intraresidual, 112 sequential and 99 long range) were complied from the nuclear Overhauser enhancement spectroscopy spectra and used in the distance geometry calculations. The core of the peptide between residues 8 and 22 assumes an alpha-helix like structure. The Cys 1-Cys 7 ring is well defined and in close association with the helix, while the C-terminal decapeptide folds back toward the core, forming a loose loop.     

Amyloid fibril formation by peptide LYS (11-36) in aqueous trifluoroethanol

Peptide LYS (11-36), derived from the beta-sheet region of T4 lysozyme, forms an amyloid fibril in aqueous trifluoroethanol (TFE) at elevated temperature. The peptide has a moderate alpha-helix content in 20 and 50% (v/v) TFE solution; large quantities of fibrils were formed after incubation at 55 degrees C for 2 weeks as monitored by a thioflavin T fluorescence assay. No fibrils were observed when the peptide initially existed predominantly as a random coil or as a complete alpha helix. Our results suggest that a moderate amount of alpha helix and random coil present in the peptide initially facilitates the fibril-formation process, but a high alpha-helix content inhibits fibril formation. Transmission electron microscopy revealed several types of fibril morphologies at different TFE concentrations. The fibrils were highly twisted and consisted of interleaved protofilaments in 50% TFE, while smooth and flat ribbonlike fibrils were found in 20% TFE. In 50% TFE, the fibril growth rate of LYS (11-36) was found to depend strongly on peptide concentration and seeding but was insensitive to solution pH and ionic strength.     

A peptide analog of the calmodulin-binding domain of myosin light chain kinase adopts an alpha-helical structure in aqueous trifluoroethanol.

A 22-residue synthetic peptide encompassing the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase was studied by two-dimensional NMR and CD spectroscopy. In water the peptide does not form any regular structure; however, addition of the helix-inducing solvent trifluoroethanol (TFE) causes it to form an alpha-helical structure. The proton NMR spectra of this peptide in 25% and 40% TFE were assigned by double quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect correlated spectroscopy spectra. In addition, the alpha-carbon chemical shifts were obtained from (1H,13C)-heteronuclear multiple quantum coherence spectra. The presence of numerous dNN(i, i + 1), d alpha N(i, i + 3), and d alpha beta(i, i + 3) NOE crosspeaks indicates that an alpha-helix can be formed from residues 3 to 20; this is further supported by the CD data. Upfield alpha-proton and downfield alpha-carbon shifts in this region of the peptide provide further support for the formation of an alpha-helix. The helix induced by TFE appears to be similar to that formed upon binding of the peptide to CaM.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin lactococcin G

The three-dimensional structures of the two peptides, lactococcin G-alpha (LcnG-alpha; contains 39 residues) and lactococcin G-beta (LcnG-beta, contains 35 residues), that constitute the two-peptide bacteriocin lactococcin G (LcnG) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles and TFE. In DPC, LcnG-alpha has an N-terminal alpha-helix (residues 3-21) that contains a GxxxG helix-helix interaction motif (residues 7-11) and a less well defined C-terminal alpha-helix (residues 24-34), and in between (residues 18-22) there is a second somewhat flexible GxxxG-motif. Its structure in TFE was similar. In DPC, LcnG-beta has an N-terminal alpha-helix (residues 6-19). The region from residues 20 to 35, which also contains a flexible GxxxG-motif (residues 18-22), appeared to be fairly unstructured in DPC. In the presence of TFE, however, the region between and including residues 23 and 32 formed a well defined alpha-helix. The N-terminal helix between and including residues 6 and 19 seen in the presence of DPC, was broken at residues 8 and 9 in the presence of TFE. The N-terminal helices, both in LcnG-alpha and -beta, are amphiphilic. We postulate that LcnG-alpha and -beta have a parallel orientation and interact through helix-helix interactions involving the first GxxxG (residues 7-11) motif in LcnG-alpha and the one (residues 18-22) in LcnG-beta, and that they thus lie in a staggered fashion relative to each other.     

Low-ultraviolet circular dichroism spectroscopy of oligopeptides 1-95 and 96-168 derived from myelin basic protein of rabbit

Myelin basic protein (MBP) is a major protein constituent of the myelin sheath of the central nervous system, where it is believed to have functional alpha-helical segments. One element of the function of the protein might be "conformational adaptability" of specific regions of its amino acid sequence, since the purified protein appears to be largely devoid of ordered structure. To pursue this question, low-ultraviolet circular dichroism (CD) spectroscopy was conducted on the sequential thrombic peptides 1-95 and 96-168 of the protein in the presence of 0-92% trifluoroethanol (TFE), a solvent known to promote stable secondary structures in polypeptides. The series of CD spectra of the oligopeptides were subjected to a computerized best-fit analysis of four peptide conformations, the alpha-helix, beta-structure, beta-turn, and nonordered form. Agreement between experimental and best-fit composite spectra was achieved when standard CD curves of peptide conformations were derived from known theoretical spectra and experimental spectra of polypeptides. In dilute buffer alone, oligopeptides 1-95 and 96-168 evidence no alpha-helix but significant beta-structure (18% and 23%, respectively), as well as a predominant, extended nonordered conformation. However, the two parts of the protein differed in conformational adaptability. From 0% to 30% TFE, 96-168 exhibited concomitant transitions to 10% helix and 32% beta-structure from the nonordered form. In contrast, in 10-30% TFE, 1-95 underwent a transition to approximately 21% helix with partial loss of beta-structure as well as nonordered form; higher concentrations of TFE (40-75%) promoted additional transitions to both helix and beta-structure (totaling 33% and 25%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.     

Chain length-function correlation of amphiphilic peptides. Synthesis and surface properties of a tetratetracontapeptide segment of apolipoprotein A-I

The segment corresponding to residues 121 to 164 of human plasma apolipoprotein A-I (apo-A-I) has been synthesized by the Merrifield solid phase method. The peptide binds to unilamellar phospholipid vesicles and to phospholipid-cholesterol mixed vesicles. The surface affinity of the peptide measured in this way indicated that the mechanism of binding is the same as that of apo A-I (144-165) and apo A-I itself. The peptide appears to be a globular monomer in a aqueous solution, with 17% alpha helix content. The peptide bound to vesicles activates lecithin:cholesterol acyltransferase: compared to apo A-I, the peptide is about 30% as efficient in the activation of cholesterol esterification and of phospholipid hydrolysis when the surface is saturated by the activator. For a variety of amphiphilic peptides and for apo A-I, the lecithin: cholesterol acyltransferase-activating ability correlates well with their alpha helix contents in 50% trifluoroethanol.     

Structure of an analog of fusion peptide from hemagglutinin

A 20-residue peptide E5 containing five glutamates, an analog of the fusion peptide of influenza virus hemagglutinin (HA) exhibiting fusion activity at acidic pH lower than 6.0-6.5 was studied by circular dichroism (CD), Fourier transform infrared, and 1H-NMR spectroscopy in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine (DPC) micelles, and phospholipid vesicles. E5 became structurally ordered at pH < or = 6 and the helical content in the peptide increased in the row: water < water/TFE < DPC approximately = phospholipid vesicle while the amount of beta-structure was approximately reverse. 1H-NMR data and line-broadening effect of 5-, 16-doxylstearates on proton resonances of DPC bound peptide showed E5 forms amphiphilic alpha-helix in residues 2-18, which is flexible in 11-18 part. The analysis of the proton chemical shifts of DPC bound and CD intensity at 220 nm of phospholipid bound E5 showed that the pH dependence of helical content is characterized by the same pKa approximately 5.6. Only Glu11 and Glu15 in DPC bound peptide showed such elevated pKas, presumably due to transient hydrogen bond(s) Glu11 (Glu15) deltaCOO- (H+)...HN Glu15 that dispose(s) the side chain of Glu11 (Glu15) residue(s) close to the micelle/water interface. These glutamates are present in the HA-fusion peptide and the experimental half-maximal pH of fusion for HA and E5 peptides is approximately 5.6. Therefore, a specific anchorage of these peptides onto membrane necessary for fusion is likely driven by the protonation of the carboxylate group of Glu11 (Glu15) residue(s) participating in transient hydrogen bond(s).     

Helix-enhancing propensity of fluoro and alkyl alcohols: influence of pH, temperature and cosolvent concentration on the helical conformation of peptides.

We have analyzed the effects of trifluoroethanol (TFE) and three other alcohols(1-propanol, 2-propanol and hexafluoro-2-propanol) on S-peptide (residues 1-20) of ribonuclease A, an analog of S-peptide (QHM-->AAA, Sa-peptide) and TC-peptide (residues 295-316) of thermolysin to assess the helix-enhancing propensity of fluoro and alkyl alcohols under different environmental conditions of cosolvent concentration, pH and temperature by circular dichroism (CD). The dependence of cosolvent concentration on helix-induction showed a plateauing effect in all cases. 1-Propanol and 2-propanol were as effective as TFE in all the three peptides. Hexafluoro-2-propanol (HFIP) was a better helix enhancer in all cases however, the relative effectiveness varied with the peptide sequence. The alcohol transitions were analyzed assuming a two-state transition. The free energy decreased linearly in the cosolvent concentration range of 0-5 m for all the three peptides. The m-value (constant of proportionality) varied between peptides but was similar for any given peptide for TFE, 1-propanol or 2-propanol. The m-values of HFIP for all three peptides was much higher compared to other cosolvents. The isothermal cosolvent helix-induction curves for the three peptides exhibited similar features of shape and character for 1-propanol, 2-propanol and TFE. The additivity of cosolvent-induced helix formation was observed for different blends of alkyl and/or fluoro cosolvents. The pH-dependence of helix formation was observed in both TFE and 1-propanol solutions for S-peptide and TC-peptide, respectively, while in Sa-peptide, which was designed to perturb the pH-effect, helix formation was unaffected. The overall results provide some insight into the mechanism of cosolvent-mediated helix-enhancement in protein segments and are likely to facilitate optimization of conditions for cosolvent usage in chemistry and biology.     

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.     

Conformational analysis by NMR and distance geometry techniques of a peptide mimetic of the third helix of the Antennapedia homeodomain.

The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well-known helix-turn-helix motif. The recognition helix penetrates the DNA major groove, gives specific protein-DNA contacts and forms direct, or water-mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42-53, where residues W48 and F49, not involved in the protein-DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well-defined secondary structure in TFE and in TFE/water (90/10, v/v) with an alpha-helix encompassing residues 4-9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted alpha-helical conformation involving residues 4-8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the alpha-helical conformation even if separated from the rest of the molecule.     

Solution structure of the osteogenic 1-31 fragment of the human parathyroid hormone

The solution conformations of a selectively osteogenic 1-31 fragment of the human parathyroid hormone (hPTH), hPTH(1-31)NH(2), have been characterized by use of very high field NMR spectroscopy at 800 MHz. The combination of the CalphaH proton and (13)Calpha chemical shifts, (3)J(NH)(alpha) coupling constants, NH proton temperature coefficients, and backbone NOEs reveals that the hPTH(1-31)NH(2) peptide has well-formed helical structures localized in two distinct segments of the polypeptide backbone. There are also many characteristic NOEs defining specific side-chain/backbone and side-chain/side-chain contacts within both helical structures. The solution structure of hPTH(1-31)NH(2) contains a short N-terminal helical segment for residues 3-11, including the helix capping residues 3 and 11 and a long C-terminal helix for residues 16-30. The two helical structures are reinforced by well-defined capping motifs and side-chain packing interactions within and at both ends of these helices. On one face of the C-terminal helix, there are side-chain pairs of Glu22-Arg25, Glu22-Lys26, and Arg25-Gln29 that can form ion-pair and/or hydrogen bonding interactions. On the opposite face of this helix, there are characteristic hydrophobic interactions involving the aromatic side chain of Trp23 packing against the aliphatic side chains of Leu15, Leu24, Lys27, and Leu28. There is also a linear array of hydrophobic residues from Val2, to Leu7, to Leu11 and continuing on to residues His14 and Leu15 in the hinge region and to Trp23 in the C-terminal helix. Capping and hydrophobic interactions at the end of the N-terminal and at the beginning of the C-terminal helix appear to consolidate the helical structures into a V-shaped overall conformation for at least the folded population of the hPTH(1-31)NH(2) peptide. Stabilization of well-folded conformations in this linear 1-31 peptide fragment and possibly other analogues of human PTH may have a significant impact on the biological activities of the PTH peptides in general and specifically for the osteogenic/anabolic activities of bone-building PTH analogues.