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 α-helical structures, whereas in more hydrophilic environments only peptide 78–108 has a helical structure. ¹H-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, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides in TFE/water were generated by distance geometry calculations. A well-defined α-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.     

Conformational mapping of a viral fusion peptide in structure-promoting solvents using circular dichroism and electrospray mass spectrometry

The N-terminal domain of human immunodeficiency virus (HIV)-1 glycoprotein 41,000 (FP; residues 1–23; NH₂-AVGIGALFLGFLGAAGSTMGARS-CONH₂) is involved in the fusion and cytolytic processes underlying viral-cell infection. Here, we use circular dichroism (CD) spectroscopy, along with electrospray ionization (ESI) mass spectrometry and tandem (MS/MS) mass spectrometry during the course of hydrogen/deuterium exchange, to probe the local conformations of this synthetic peptide in two membrane mimics. Since amino acids that participate in defined secondary structure (i.e., α-helix or β-sheet) exchange amido hydrogens more slowly than residues in random structures, deuterium exchange was combined with CD spectroscopy to map conformations to specific residues. For FP suspended in the highly structure-promoting solvent hexafluoroisopropanol (HFIP), CD spectra indicated high α-helix and disordered structures, whereas ESI and MS/MS mass spectrometry indicated that residues 5–15 were α-helical and 16–23 were disordered. For FP suspended in the less structure-promoting solvent trifluoroethanol (TFE), CD spectra showed lower α-helix, with ESI and MS/MS mass spectrometry indicating that only residues 9–15 participated in the α-helix. These results compare favorably with previous two-dimensional nuclear magnetic resonance studies on the same peptide. Proteins Suppl. 2:38–49, 1998. © 1998 Wiley-Liss, Inc.     

Conformational Analysis of Neuropeptide Y Segments by CD,NMR Spectroscopy and Restrained Molecular Dynamics

Neuropeptide Y (NPY), a peptide amide comprising 36 residue has been shown to act as a potent vasoconstrictor. In order to shed light on the structural requirements for the biological activities with respect to the different prerequisites for affinity to the NPY receptor subtypes Y₁ and Y₂, in the present study the syntheses and conformational analyses of two C-terminal segments, NPY(18–36) and NPY(13–36), are described. The results obtained by CD measurements, two-dimensional NMR spectros copy and a conformational refinement of the NMR-derived structure by molecular mechanics simulations support the findings of previously published structure –activity relationship studies for biologically active and selective compounds. In particular, the α-helical conformation as well as an appropriate exposure of the side chains of the critical C-terminal dipeptide within NPY(18–36) are in agreement with the prerequisites proposed for Y₂ receptor binding of that segment.     

Conformation of four peptides corresponding to the α-helical segments of human GM–CSF

The conformation of segments corresponding to the four α-helical stretches found in human granulocyte-macrophage colony-stimulating factor was studied in water solution in the presence of different amounts of 2,2,2-trifluoroethanol (TFE). The CD spectra reveal the onset of secondary structure upon addition of TFE. The final amount of helical conformation varies among the four peptides. In all cases, the conformational transition is complete before 50% TFE (v/v). ¹H-NMR studies were conducted at this solvent composition, leading to the assignment of all the resonances and to the definition of the secondary structure for all four fragments. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci.3: 336–346 No. of Figures: 12. No. of Tables: 5. No. of References: 25     

Conformational mapping of the N-terminal peptide of HIV-1 gp41 in membrane environments using C-enhanced Fourier transform infrared spectroscopy

The N-terminal domain of HIV-1 glycoprotein 41?000 (FP; residues 1-23; AVGIGALFLGFLGAAGSTMGARSCONH₂) participates in fusion processes underlying virus-cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, ¹²C-Fourier transform infrared (FTIR) spectroscopy indicated the following α-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes∼hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). ¹²C-FTIR spectra also showed disordered FP structures in these environments, along with substantial β-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with ¹³C-carbonyl at multiple sites. Combining these ¹³C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: α-helix (residues 3-16) and random and β-structures (residues 1-2 and residues 17-23). Additional ¹³C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the α-helix extends over residues 1-16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, ¹³C-FTIR spectroscopy showed α-helical conformations for the central core of FP (residues 5-15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel β-structure. FP at low loading in ghosts probably inserts deeply as an α-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, β-sheet. In future studies, ¹³C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.     

A new approach to secondary structure evaluation: Secondary structure prediction of porcine adenylate kinase and yeast guanylate kinase by CD spectroscopy of overlapping synthetic peptide segments

A new approach for evaluating the secondary structure of proteins by CD spectroscopy of overlapping peptide segments is applied to porcine adenylate kinase (AK1) and yeast guanylate kinase (GK3). One hundred seventy-six peptide segments of a length of 15 residues, overlapping by 13 residues and covering the complete sequences of AK1 and GK3, were synthesized in order to evaluate their secondary structure composition by CD spectroscopy. The peptides were prepared by solid phase multiple peptide synthesis method using the 9-fluorenylmethoxycarbonyl/tert-butyl strategy. The individual peptide secondary structures were studied with CD spectroscopy in a mixture of 30% trifluoroethanol in phosphate buffer (pH 7) and subsequently compared with x-ray data of AK1 and GK3. Peptide segments that cover α-helical regions of the AK1 or GK3 sequence mainly showed CD spectra with increasing and decreasing Cotton effects that were typical for appearing and disappearing α-helical structures. For segments with dominating β-sheet conformation, however, the application of this method is limited due to the stability and clustering of β-sheet segments in solution and due to the difficult interpretation of random-coiled superimposed β-sheet CD signals. Nevertheless, the results of this method especially for α-helical segments are very impressive. All α-helical and 71% of the β-sheet containing regions of the AK1 and GK3 could be identified. Moreover, it was shown that CD spectra of consecutive peptide content reveal the appearance and disappearance of α-helical secondary structure elements and help localizing them on the sequence string. © 1997 John Wiley & Sons, Inc. Biopoly 41: 213–231, 1997     

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.     

Conformational aspects of gastrin-related peptides: A circular dichroism study

The conformational properties of a series of gastrin-related peptides in aqueous solution and in 2,2,2-trifluoroethanol (TFE) have been investigated by CD measurements. In aqueous solution the peptides Leu³²-HG-34 (human big gastrin), Nle¹⁵-HG-17 (human little gastrin), and Nle¹¹-HG-13 assume a random-coil structure in the pH range 3–7. In TFE the three hormones fold into partially ordered structures, consisting of mixtures of α-helix, β-form and random coil. Comparison with the CD properties of the shorter gastrin peptides HG-4 (tetragastrin), N^α-Boc-HG-5 (pentagastrin), and HG-7 (heptagastrin) indicates that the biologically important C-terminal sequence Trp-Met-Asp-Phe-NH₂ in TFE does not maintain the same geometry upon elongation of the chain at the N-terminus from 4 to 34 residues. Thus, the various conformations in solution of the gastrin peptides examined do not provide a structural explanation for their very similar biological activity. Therefore, we hypothesize that the C-terminal tetrapeptide amide folds into an “active” structure only upon interaction with the receptor.     

Solution structure of the fifth and sixth transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the fifth and sixth transmembrane segments of the bovine mitochondrial oxoglutarate carrier (OGC) and of the hydrophilic loop that connects them were studied by CD and NMR spectroscopies. Peptides F215-R246, W279-K305 and P257-L278 were synthesized and structurally characterized. CD data showed that at high concentrations of TFE and SDS all peptides assume α-helical structures. ¹H-NMR spectra of the three peptides in TFE/water were fully assigned and the secondary structures of the peptides were obtained from nuclear Overhauser effects, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides were generated by distance geometry calculations. A well-defined α–helix was found in the region L220-V243 of peptide F215-R246 (TMS-V), in the region P284-M303 of peptide W279-K305 (TMS-VI) and in the region N261-F275 of peptide P257-L278 (hydrophilic loop). The helix L220-V243 exhibited a sharp kink at P239, while a little bend around P291 was observed in the helical region P284-M303. Fluorescence studies performed on peptide W279-K305, alone and together with other transmembrane segments of OGC, showed that the W279 fluorescence was quenched upon addition of peptide F215-R246, but not of peptides K21-K46, R78-R108 and P117-A149 suggesting a specific interaction between TMS-V and TMS-VI of OGC.     

A high-resolution 13C-nmr study of collagenlike polypeptides and collagen fibrils in solid state studied by the cross-polarization–magic angle-spinning method. Manifestation of conformation-dependent 13C chemical shifts and application to conformational characterization

We have recorded high-resolution ¹³C-nmr spectra of collagen fibrils in the solid state by the cross-polarization–magic-angle-spinning(CP–MAS)method and analyzed the spectra with reference to those of collagenlike polypeptides. We used two kinds of model polypeptides to obtain reference ¹³C chemical shifts of major amino acid residues of collagen (Gly, Pro, Ala, and Hyp): the 3₁-helical polypeptides [(Gly)_nII, (Pro)_nII, (Hyp)_n, and (Ala? Gly? Gly)_nII], and the triple-helical polypeptides [(Pro? Gly? Pro)_n and (Pro? Ala? Gly)_n]. Examination of the ¹³C chemical shifts of these polypeptides, together with our previous data, showed that the ¹³C chemical shifts of individual amino acid residues are the same, within experimental error (±0.5 ppm), among different polypeptides with different primary sequences, if the conformations are the same. We found that the ¹³C chemical shifts of Ala residues of the 3₁-helical (Ala? Gly? Gly)_n and triple-helical (Pro? Ala? Gly)_n are significantly displaced, compared with those of the α-helix, β-sheet, and silk I form, and can be utilized as excellent probes to examine conformational features of collagen-like polypeptides. Further, the ¹³C chemical shifts of Gly and Pro residues in the triple-helical polypeptides are substantially displaced from those found in (Gly)_nII and (Pro)_nII of the 3₁-helix, reflecting further conformational change from the 3₁-helix to the supercoiled triple helix. In particular, the ¹³C chemical shifts of Gly C ? O carbons of the triple-helical polypeptides are substantially displaced upfield (4.1–5.1 ppm), with respect to those of the 3₁-helical polypeptides. These displacements are interpreted by that Gly C ? O of the former is not involved in NH …? O ? C hydrogen bonds, while this carbon of the latter is linked by these kinds of hydrogen bonds. On the basis of these ¹³C chemical shifts, as reference data for the collagenlike structure, we were able to assign the ¹³C-nmr peaks of Gly, Ala, Pro, and Hyp residues of collagen fibrils, which are in good agreement with the values expected from the model polypeptides mentioned above. We also discuss a plausible conformational change of collagen fibrils during denaturation.     

1H-nmr study of protected methionine homo-oligopeptides in helix-supporting environment

¹H-nmr spectra for a series of Boc-L -(Met)_n-OMe (n = 2–9) homo-oligopeptides have been observed in the helix-supporting solvent trifluoroethanol (TFE) at millimolar concentrations. Interfering solvent peaks were eliminated using two decoupling frequencies to selectively remove the methylene and hydroxyl protons of the solvent. Comparisons with specifically α-deuterated homo-oligopeptides gave complete assignments of the NH region of the Boc-Met_n-OMe oligomers up to the heptapeptide. Analysis of chemical shifts, coupling constants, and temperature dependence of chemical shifts suggests that up to the hexapeptide, similar structures exist in deuterochloroform and TFE. In contrast, nmr parameters at the heptapeptide for several internal residues differ in these solvents. These results suggest that a C₇ to α-helix transition may occur in TFE as the chain length of the methionine oligopeptides increases.     

Stabilizing effects of 2-methylalanine residues on β-turns and α-helices

¹³C-, ¹H-nmr, CD, and x-ray crystallography revealed β-turns of type III for Boc-Gly-L-Ala-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 3₁₀-helix for Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe; and antiparallel arranged α-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe. An N-terminal rigid α-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The α-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole–dipole interactions of parallel and antiparallel arranged α-helices within oligomeric structures.     

Assignment of the helical structure in neuropeptide Y by HPLC studies of methionine replacement analogues and 1H-NMR spectroscopy

The HPLC retention behavior of three complete single methionine and methionine sulfoxide replacement sets of two 18-mer model peptides and neuropeptide Y (NPY) were investigated. All peptides were prepared by multiple solid-phase peptide synthesis. Plotting the retention time differences between methionine and methionine sulfoxide analogues vs the position of replacement shows that potentially α-helical peptides become helical on binding during reversed-phase high performance liquid chromatography. In the case of an amphipathic α-helix, the retention time differences change periodically with a 3–4 repeat pattern, which allow the location of amphipathic helical structures. Replacements in nonamphipathic α-helical domains cause local preferential binding areas and lead to sequence-dependent retention time profiles. Methionine replacement studies of NPY suggest an unstructured or extended conformation from Tyr¹ to Ala¹² connected to a well-defined amphipathic α-helix from Pro¹³ to Arg³⁵. The assignment is confirmed by comparison of nuclear Overhauser effects based two-dimensional ¹H-nmr spectroscopy and utilization of the C^αH shift index method in 50% trifluoroethanol/50% water. © 1996 John Wiley & Sons, Inc.     

Conformational studies of the α-helical proteins from wool keratin by c.d.

The conformation and conformational change of wool keratin $\text{S-}\text{carboxymethylated}$ low-sulphur proteins (SCMKA), which are α-helical fibrous proteins, have been investigated in aqueous solution by means of c.d. Comparisons of various methods proposed for c.d. analysis of protein secondary structure are made using least-squares curve-fitting of the observed c.d. spectra of SCMKA with a linear combination of the corresponding reference spectra of secondary structures. It has been found that (i) the most satisfactory results are obtained with the method¹³ which takes into account the β-turn contribution: (ii) SCMKA is 52–54% α-helical in water and has little β-form, (iii) the addition of n-propanol produces, even at higher concentrations of n-propanol, little chnage in spectra with respect to helical character in water; (iv) SCMKA undergoes a thermally-induced conformational transition from α-helix to random coil around 50 C; and (v) $\text{S-}\text{aminoethylated}$ low-sulphur proteins with positively charged protecting groups are /_~50% α-helical in water, which is similar to SCMKA, showing that the protecting groups introduced in the low-sulphur proteins are little effect upon their conformation in water     

Conformation and properties of DNA–(Lys33, Leu67)100–(Orn)20 complex: A nucleohistone model

The synthesis and characterization of the block copolypeptide (Leu⁶⁷, Lys³³)₁₀₀Orn₂₀, a synthetic model of histone, are reported. In neutral aqueous solutions, 80% of the etheropolypeptide block assumes an α-helical conformation, whereas the polyornithine block is in a random-coil conformation. In the association complexes with DNA, melting and titration experiments, as well as CD results, indicate that the polyornithine block interacts with DNA, whereas at least 2/3 of the lysine residues of the (Leu, Lys) moiety are excluded from the direct binding with DNA. CD spectra of the association complexes reveal significant differences from those obtained with DNA–polyornithine and DNA–polylysine complexes but substantial similarities with CD spectra of native and reconstituted nucleohistones. In contrast to DNA–polyornithine complexes, the CD spectra of the ternary complexes, copolypeptide–DNA–ethidium bromide, indicate a strong reduction of the dye intercalation. The low-angle x-ray diffraction pattern, reminiscent of that of chromatin, reveals the presence of a superstructure in these complexes. The results obtained are discussed in connection with the expected structural features of the model.     

Vibrational and chiroptical spectroscopic characterization of γ‐turn model cyclic tetrapeptides containing two β‐Ala residues

The optical spectroscopic characterization of γ‐turns in solution is uncertain and their distinction from β‐turns is often difficult. This work reports systematic ECD and vibrational circular dichroism (VCD) spectroscopic studies on γ‐turn model cyclic tetrapeptides cyclo(Ala‐β‐Ala‐Pro‐β‐Ala) ( 1 ), cyclo(Pro‐β‐Ala‐Pro‐β‐Ala) ( 2 ) and cyclo(Ala‐β‐Ala‐Ala‐β‐Ala) ( 3 ). Conformational analysis performed at the 6‐31G(d)/B3LYP level of theory using an adequate PCM solvent model predicted one predominant conformer for 1‐3 , featuring two inverse γ‐turns. The ECD spectra in ACN of 1 and 2 are characterized by a negative n→π* band near 230 nm and a positive π→π* band below 200 nm with a long wavelength shoulder. The ECD spectra in TFE of 1‐3 show similar spectra with blue‐shifted bands. The VCD spectra in ACN‐d₃ of 1 and 2 show a +/?/+/? amide I sign pattern resulting from four uncoupled vibrations in the case of 1 and a sequence of two positive couplets in the case of 2 . A ?/+/+/? amide I VCD pattern was measured for 3 in TFE‐d₂. All three peptides give a positive couplet or couplet‐like feature (+/?) in the amide II region. VCD spectroscopy, in agreement with theoretical calculations revealed that low frequency amide I vibrations (at ～1630 cm^?1 or below) are indicative of a C₇ H‐bonded inverse γ‐turns with Pro in position 2, while γ‐turns encompassing Ala absorb at higher frequency (above 1645 cm^?1). Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Proline-induced constraints in alpha-helices

The disrupting effect of a prolyl residue on an α-helix has been analyzed by means of conformational energy computations. In the preferred, nearly α-helical conformations of Ac-Ala₄-Pro-NHMe and of Ac-Ala₇-Pro-Ala₇-NHMe, only the residue preceding Pro is not α-helical, while all other residues can occur in the α-helical A conformation; i.e., it is sufficient to introduce a conformational change of only one residue in order to accommodate proline in a distorted α-helix. Other low-energy conformations exist in which the conformational state of three residues preceding proline is altered considerably; on the other hand, another conformation in which these three residues retain the near-α-helical A-conformational state (with up to 26° changes of their dihedral angles ? and ψ, and a 48° change in one ω from those of the ideal α-helix) has a considerably higher energy. These conclusions are not altered by the substitution of other residues in the place of the Ala preceding Pro. The conformations of the peptide chain next to prolyl residues in or near an α-helix have been analyzed in 58 proteins of known structure, based on published atomic coordinates. Of 331 α-helices, 61 have a Pro at or next to their N-terminus, 21 have a Pro next to their C-terminus, and 30 contain a Pro inside the helix. Of the latter, 16 correspond to a break in the helix, 9 are located inside distorted first turns of the helix, and 5 are parts of irregular helices. Thus, the reported occurrence of prolyl residues next to or inside observed α-helices in proteins is consistent with the computed steric and energetic requirements of prolyl peptides.     

Circular dichroism of the complex between N-N′-diethylpseudoisocyanine and sodium poly(L-glutamate) in aqueous solutions

In the presence of sodium poly(L -glutamate) at pH = 7.5 the dye pseudoisocyanine in dilute aqueous solution (C_d = 1.10 × 10^?5 M) and low P/D values exhibits an absorption spectrum with a very sharp red-shifted J-band. Under the same conditions circular dichroism (CD) in the visible region is observed with an extremely sharp peak at the position of the J-band. At pH = 4.6, where the polypeptide is in the α-helix conformation, no such J-band is observed and no CD spectrum can be detected at the same P/D values. CD spectra in the uv range demonstrate that the occurrence of the dye–polypeptide complex which gives rise to the J-band at slightly alkaline pH is not accompanied by a conformational transition of the polypeptide towards the α-helical form.     

Design and synthesis of novel nonpolar host peptides for the determination of the 310- and α-helix compatibilities of α-amino acid buildig blocks: An assessment of α,α-disubstituted glycines

The present work describes three novel nonpolar host peptide sequences that provide a ready assessment of the 3₁₀- and α-helix compatibilities of natural and unnatural amino acids at different positions of small- to medium-size peptides. The unpolar peptides containing Ala, Aib, and a C-terminal p-iodoanilide group were designed in such a way that the peptides could be rapidly assembled in a modular fashion, were highly soluble in solvent mixtures of triflouroethanol and H₂O for CD- and two-dimensional (2D) nmr spectroscopic analyses, and showed excellent crystallinity suited for x-ray structure analysis. To validate our approach we synthesized 9-mer peptides 79a–96 (Table IV), 12-mer peptides 99–110c (Table V), and 10-mer peptides 120a–125d and 129–133 (Table VI and Scheme 8) incorporating a series of optically pure cyclic and open-chain (R)- and (S)-α,α-disubstituted glycines 1–10 (Figure 2). These amino acids are known to significantly modulate the conformations of small peptides. Based on x-ray structures of 9-mers 79a, 80, and 87 (Figures 4–7), 10-mers 124c, 131, and 132 (Figures 9–12), and 12-mer peptide 102b (Figure 13), CD spectra of all peptides recorded in acidic, neutral, and basic media and detailed 2D-nmr analyses of 9-mer peptide 86 and 12-mer 102b, several interesting conformational observations were made. Especially interesting results were obtained using the convex constraint CD analysis proposed by Fasman on 9-mer peptides 79a–d, 80, 81, 86, and 87, which allowed us to determine the relative content of 3₁₀- and α-helical conformations. These results were fully supported by the corresponding x-ray and 2D-nmr analyses. As a striking example we found that the (S)- and (R)-β-tetralin derived amino acids (R)- and (S)-1 show excellent α-helix stabilisation, more pronounced than Aib and Ala. These novel reference peptide sequences should help establish a scale for natural and unnatural amino acids concerning their intrinsic 3₁₀- and α-helix compatibilities at different positions of medium-sized peptides and thus improve our understanding in the folding processes of peptides. © 1997 John Wiley & Sons, Inc. Biopoly 42: 575–626, 1997     

Purification, molecular cloning, and antimicrobial activity of peptides from the skin secretion of the black-spotted frog, Rana nigromaculata

Antimicrobial peptides from a wide range of amphibian species, especially frogs of the genus Rana, have been characterised and are potential therapeutic agents. Here we describe the isolation, purification, and structural and biological characterisation of three novel antimicrobial peptides from the skin secretions of the black spotted frog, Rana nigromaculata, from Northeastern China. The peptides were identified as belonging to two known families: the temporin, which was first identified in R. nigromaculata from China, and the brevinin-2. Temporin-1RNa and temporin-1RNb both containing three positive charges and have a high potency against microorganisms (MIC: 3.13–8.3 μM against Gram-positive bacteria, 12.5–25.0 μM against Gram-negative bacteria, and 6.25–12.5 μM against Candida albicans) and a high haemolytic activity against human erythrocytes (HC₅₀: 100–150 μM). Brevinin-2RNa contains a single intra-disulphide bridge at the C-terminus that is active towards the tested Gram-positive bacteria but is not active against E. coli and P. aeruginosa. The cDNAs encoding three novel peptide precursors were also subsequently cloned from an R. nigromaculata skin cDNA library and sequenced. The precursors contain 58–72 amino acid residues, which include a conserved signal peptide, acidic propeptide, and the mature temporin-1RNa, temporin-1RNb and brevinin-2RNa. The CD spectra of temporin-1RNa and temporin-1RNb in water, 30 mM SDS and 50 % trifluoroethanol (TFE) indicated that both peptides adopted an aperiodic structure in water and an organised structure with an α-helical conformation in TFE and SDS solution. The conformational transition induced by TFE or SDS reflects the potential ability of temporin-1RNa and temporin-1RNb to interact with anionic membranes.