Block sequential polypeptides of L-alanine and glycine with D, L-glutamic acid

Sequential polypeptides of L -alanine(A) and glycine(G), which were incorporated between two blocks of poly(D ,L -glutamic acid) (DL), were synthesized by applying Merri-field's solid-phase method. On the basis of optical rotatory dispersion criteria, DL(A)₃₈-DL was found to assume the α-helix in the whole range of the water-methanol system; whereas other block sequential polypeptides were found to assume the random-coiled conformation in water and partly the α-helix at the high methanol content. The stability of the α-helix decreased in the order: DL(A)₃₈DL, DL(A₂G)₁₀DL, DL(A₂G)₆DL, and DL(A₃G)₇DL. This phenomenon may be explained in terms of the dependence of hydrophobic bonding between the C₃H group of the ith L -alanine regularly arranged on the surface of the α-helix and the C₂H group of the (i + 3)th residue on whether the residue is alanine or glycine. The role which the methanol plays in stabilizing the α-helix is also discussed.     

Basic polypeptides as histone models. Synthesis, conformation, and interaction with DNA of statistical copolymers (Lys x,Ala y)n.

Statistical copolymers (Lys^x,Ala^y)_n were synthesized by copolymerization of N-carboxyanhydrides of L -amino acids. The conformation of copolymers in aqueous solutions was investigated using circular dichroism (CD). Calculations based on the CD data showed that polymers (Lys^x,Ala^y)_n can exhibit a random conformation, an α-helix, and a β-structure in various ratios. CD spectra of complexes of copolymers with DNA prepared by gradual dialysis from a high ionic strength to 0.15 M NaCl can be correlated with the copolymer conformation in medium and high ionic strength. For copolymers forming an α-helix and β-structure, these spectra show resemblance with similar spectra of complexes of those histones that are able to exhibit ordered conformations.     

13C-nmr chemical shift and conformation of L-alanine residues incorporated into poly(β-benzyl L-aspartate) in the solid state

¹³C-nmr spectra of poly(β-benzyl L-aspartate) containing ¹³C-enriched [3-¹³C]L -alanine residues in the solid state were recorded by the cross polarization–magic angle spinning method, in order to elucidate the conformation-dependent ¹³C chemical shifts of L -alanine residues taking various conformations such as the antiparallel β-sheet, the right-handed α-helix, the left-handed α-helix, and the left-handed ω-helix forms obtained by appropriate treatment. The latter two conformations for L -alanine residues are achieved when L -alanine residues are incorporated into poly(β-benzyl L -aspartate). We found that the alanine C_β carbon show significant ¹³C chemical shift displacement depending on conformational change, and gave the ¹³C chemical shift values at about 17 ppm for the left-handed ω-helix, 14 ppm for the left-handed α-helix, 15.5 ppm for the right-handed α-helix, and 21.0 ppm for the antiparallel β-sheet relative to tetramethylsilane.     

Vibrational CD of polypeptides. X. A study of alpha-helical oligopeptides in solution

We have measured the vibrational CD (VCD) of a series of heterooligopeptides—o-nitrophenylsulfenyl(L -Met-L -Met-L -Leu) _n-OEt, n = 6,8,10,11–in the amide A, I, and II regions. These spectra are identical in shape and magnitude, within our signal to noise limits. The VCD in each band are of exactly the shape expected for a right-handed α-helix and imply that VCD of the polypeptide α-helix is relatively unaffected by chain length down to the 18-subunit level.     

Conformation and aggregation of bovine myelin proteins

CD and PMR spectra were obtained on three major protein fractions of bovine CNS myelin: the basic A-1 protein, the Folch-Lees proteolipid apoprotein (APL), and the Wolfgram proteolipid protein (WPP). Most PMR peaks of the A-1 broadened on going from D₂O to salt solutions or to 100% 2-Chloroethanol (2-CE). CD spectra showed no α-helix in water or salt solutions, but showed 42% in 2-CE. The APL showed no PMR in D₂O, but did show aromatic amino acid peaks in 1.5% SDS. CD spectra showed 37% α-helix in both cases. The PMR of the WPP in 1.5% SDS showed aromatic amino acids, and the CD showed <20% α-helix. All three proteins showed sharp PMR spectra in trifluoroacetic acid with α-CH chemical shifts characteristic of random coils. It was concluded that the A-1 and the APL aggregate.     

Folding simulations of three proteins having all α-helix,all β-strand and α/β-structures

We performed folding simulations of three proteins using four force fields, AMBER parm96, AMBER parm99, CHARMM 27 and OPLS-AA/L, in order to examine the features of these force fields. We studied three proteins, protein A (all α-helix), cold-shock protein (all β-strand) and protein G (α/β-structures), for the folding simulations. For the simulation, we used the simulated annealing molecular dynamics method, which was performed 50 times for each protein using the four force fields. The results showed that the secondary-structure-forming tendencies are largely different among the four force fields. AMBER parm96 favours β-bridge structures and extended β-strand structures, and AMBER parm99 favours α-helix structures and 3₁₀-helix structures. CHARMM 27 slightly favours α-helix structures, and there are also π-helix and β-bridge structures. OPLS-AA/L favours α-helix structures and 3₁₀-helix structures.     

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     

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.     

Exploring the peptide 310-helix ⇆ α–helix equilibrium with double label electron spin resonance

Over the last several years we have used spin labeling as a means for exploring the structure of helical peptides. Two nitroxide labels are engineered into a peptide sequence and distances are ranked with electron spin resonance (ESR). We have found that there is a significant amount of 3₁₀–helix in 16–residue model peptides containing only L –amino acids. This review covers several facets of the methodology including spin labeling strategy, interpretation of ESR spectra and the influence of molecular dynamics on the spectral line shapes. Also covered are recent findings of a length–dependent 3_l0-helix → α-helix transition and the role of Arg⁺ in the stabilization of specific helix structures. © 1994 John Wiley & Sons, Inc.     

Raman spectra of copoly(D,L-alanines)

Raman spectra in the region 1000–150 cm^?1 were measured for copoly(D ,L -alanines) with the D -residue contents, 3, 7, 10, and 20%, and compared with the spectrum of the α-helical poly-L -alanine. The 532- and 378-cm^?1 peaks were assigned to the L -residues with a right-handed α-helix-like local conformation or to the D -residues with a left-handed α-helix-like local conformation. From the intensity of the latter peak the contents of these local conformations were estimated as a function of the D -residue contents for the copolymers. The 264-cm^?1 peak, which has been assigned to the breathing vibration of the α-helical poly-L -alanine, shows a marked decrease in its intensity upon the introduction of the D residues. This result suggests that the overall deformation vibration of the α-helix arises from rather long sequences of the L - and D -alanine residues with the α-helical conformation and that the intensity of this vibration depends on the content of these sequences in the copolymers.     

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.     

Critical Main-Chain Length for Conformational Conversion From 310-Helix to α-Helix in Polypeptides

Abstract

To assess the minimal peptide length required for the stabilization of the a-helix relative to the 3₁₀-helix in Aib-rich peptides, we have solved the X-ray diffraction structures of the terminally blocked sequential hexa- and octapeptides with the general formula -(Aib-L-Ala)_n-(n = 3 and 4, respectively). The hexapeptide molecules are completely 3₁₀-helical with four 1 ← 4 intramolecular N-H … O=C H-bonds. On the other hand, the octapeptide molecules are essentially α-helical with four 1 ← 5 H-bonds; however, the helix is elongated at the N-terminus, with two 1 ← 4 H-bonds, giving these molecules a mixed α/3₁₀-helical character. In both compounds the right-handed screw sense of the helix is dictated by the presence of the Ala residues of L-configuration. This study represents the first experimental proof for a 3₁₀ →α-helix conversion in the crystal state induced by peptide backbone lengthening only.     

The measurement of transmembrane helices by the deconvolution of CD spectra of membrane proteins: A review

The interpretation of the CD spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as x-ray or nmr data. Therefore, these methods are inappropriate for a CD data base whose secondary structures are unknown, as in the case of the membrane proteins. The Convex Constraint Analysis algorithm [A. Perczel, M. Hollósi, G. Tusnády, and G. D. Fasman (1991) Protein Engineering, Vol. 4, 669–679], on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived “pure” CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of α-helices (the α-helix in the soluble domain and the α_T-helix, for the transmembrane α-helix), a β-pleated sheet, a class C-like spectrum related to β-turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the α_T-helix was characterized by a positive red-shifted band in the range 195–200 nm (+95,000 deg cm² dmol^?l), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222 nm band (?50,000 and ?60,000 deg cm² dmol^?1, respectively) in comparison with the regular α-helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of + 70,000, ?30,000, and ?30,000 deg cm² dmol^?1, respectively. © 1994 John Wiley & Sons, Inc.     

Conformational studies on poly(N-δ-trimethyl-l-ornithine)

$\text{Poly}\text{(N-δ-}\text{trimethyl-}\text{l}\text{-ornithine}$), (Me₃Orn)_n, is usually not able to attain the α-helical conformation in aqueous solution independent of its pH value; however, it becomes α-helical at low concentrations of sodium perchlorate over a wide pH range according to the circular dichorism (c.d.) spectra. Cl^?, SO₄^2? and H₂PO₄^? do not induce α-helix formation. One can conclude that a distinct topology of the anions bound by the side chains is responsible for the α-helix-inducing effect of some water-structure-breaking anions such as perchlorate. This means that the anions are inserted between the ?N⁺ of the side groups shielding the positive charges repelling one another. The insertion of the anions requires that the water molecules surrounding the ions can be stripped off, which is easily possible if they are water-structure-breaking ones. At higher perchlorate concentrations, the c.d. spectrum changes. It is characterized by a negative shoulder near 208 nm and a pronounced minimum at ≈ 226 nm. With increasing temperature, the c.d. spectrum of the α-helix occurs. Finally the α-helix undergoes a conformational change to the random coil. The apparent transition enthalpy ΔH_vH is remarkably lower than that of the homologue (Me₃Lys)_n, obviously due to a lower cooperativity of the transition. In contrast to poly(l-ornithine), (Orn)_n, the c.d. spectrum of (Me₃Orn)_n remains almost unchanged after adding anionic surfactants such as sodium octyl sulphate (SOS) or sodium dodecyl sulphate (SDS). In organic solvents like methanol or isopropanol, in contrast to (Orn)_a and (Lys)_n, no α-helix formation occurs. However, in mixtures of these alcohols or dioxane with water, α-helix formation is induced by perchlorate, as in pure water. The thermal stability of the α-helix in these systems is increased.     

Vibrational analysis of peptides,polypeptides, and proteins. XVIII. Conformational sensitivity of the α-helix spectrum: αI- and αII-Poly(L-alanine)

The α_II-helix (? = ?70.47°, ψ = ?35.75°) is a structure having the same n and h as the (standard) α_I-helix (? = ?57.37°, ψ = ?47.49°). Its conformational angles are commonly found in proteins. Using an improved α-helix force field, we have compared the vibrational frequencies of these two structures. Despite the small conformational differences, there are significant predicted differences in frequencies, particularly in the amide A, amide I, and amide II bands, and in the conformation-sensitive region below 900 cm^?1. This analysis indicates that α_II-helices are likely to be present in bacteriorhodopsin [Krimm, S. & Dwivedi, A. M. (1982) Science 216 , 407–408].     

Secondary structure of peptides. 4: 13C-Nmr CP/MAS investigation of solid oligo- and poly(L-alanines)

Primary and tertiary amine-initiated polymerizations of L -alanine-N-carboxyanhydride (L -Ala-NCA) were conducted at 20 or 100°C in a variety of solvents. The 75.5-MHz ¹³C-nmr CP/MAS spectra of the resulting poly(L -alanines) revealed that all samples contain both α-helix and pleated-sheet structures. Depending on the reaction conditions the α-helix content varied between ca. 1 and 99%. Reprecipitation from aprotic nonsolvents does not change the α-helix/β-sheet ratio, indicating that this ratio is thermodynamically controlled. Since relatively large amounts of oligopeptides of degree of polymerization (DP ) 4–6 can be extracted by means of acetic acid, it is concluded that (a) most poly(L -alanines) possess a bimodal molecular weight distribution, (b) the oligopeptide fraction with DP ? 11 is responsible for the β-sheet fraction of all samples, and (c) the two-stage crystal growth proposed by Komoto and Kawai is not correct. Solubilizing initiators such as poly(ethylene oxide) NH₂ prevent the precipitation of oligoalanine and, thus, the formation of a β-sheet structure. ¹³C-nmr CP/MAS measurements also show that tri- and tetra-L -alanines form insoluble β-sheet structures.     

Secondary structure perturbations in salt-induced protein precipitates

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na₂SO₄, were studied for twelve different proteins. α-helix and β-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of β-sheet structures with a concomitant loss of α-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of α-helix and β-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting β-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in α-helix content were influenced by the initial helical content and the dipole moment. The enhanced β-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.     

Conformational properties of the proline region of porcine neuropeptide Y by CD and 1H-NMR spectroscopy

We synthesized porcine neuropeptide Y (pNPY) N-terminal fragments by solid-phase synthesis techniques and analyzed them for solution Conformational properties by CD and ¹H-nmr spectroscopy. The analogues pNPY_1–9 and pNPY_1–14 displayed CD spectra indicative of random structures and showed no evidence for induced α-helical structures in trifluoroethanol (TFE) up to 50%. However, the CD spectra of pNPY_1-9 suggested a Conformational shift in tetrahydrofuran. Although in aqueous solution the CD spectra of pNPY_1–21 indicated random structures with induction of only a small percentage of α-helix in aqueous TFE, pNPY_1-25 displayed 13% a-helical structure in aqueous solution that increased to 40 and 41% by the addition of TFE and methanol, respectively. The nmr spectra of pNPY_1-9 and the proline region of pNPY_1–25 indicated extended structures with all-trans conformers at Pro5 and Pro8 for pNPY_1–9 and at Pro5, Pro8, and Pro13 for pNPY_1–25; in each case the Tyrl-Pro2 amide bond was in both cis and trans conformations. However, observed nuclear Overhauser effect correlations and UN exchange experiments indicated an α-helical segment in pNPY_1–25 initiated by Pro 13 and extending from residues 14 to 25. Thus, the N-terminal polyproline region of NPY has no propensity to fold into a regular secondary structure, although Pro 13 is a helix initiator, a result consistent with the proposed role of this amino acid in the NPY structural model. © 1995 John Wiley & Sons, Inc.     

Effects of salts on the nonequivalent stability of the α-helices of isomeric block copolypeptides

The stability of the α-helices of isomeric block copolypeptides is nonequivalent, as reported previously. In order to explore the origin of the nonequivalence, the stability of α-helix of two block copolypeptides, (L -Ala)₂₀-(L -Glu)₂₀-(L -Phe) (designated as AEF) and (L -Glu)₂₀-(L -Ala)₂₀-(L -Phe) (EAF), in aqueous solution was investigated as a function of pH, temperature, and salt concentration by the measurement of the α-helical content using CD at 223 nm. The transition temperature, T_m, as a measure of the stability of the α-helix, decreased with increasing the salt concentration for EAF, while T_m increased for AEF. The results indicate that electrostatic interactions affect the nonequivalence of such helical stability. Thermodynamic quantities, ΔG, ΔH, and ΔS, of the thermal transition from random coil to α-helix were obtained by applying the curve-fitting method to the data. The major contribution to the effects of salts seems to be the entropic term, not the enthalpy term. This is unexpected, since the salt ions would weaken electrostatic interactions between ionized groups and the dipole along the helical axis, which affect the enthalpy term. In addition, the dependence of the electrostatic effect on the salt concentration is different for EAF and AEF. There fore, the nonequivalence cannot be accounted for by only the electrostatic effect, suggesting that it originates from some intrinsic property of the α-helix.     

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.