Synthesis and conformational study of sequential polypeptides, (L-ala-L-val-gly)n and (L-val-L-ala-gly)n.

As an approach for elucidating the role of sequences of amino acids in protein structures, model polypeptides having the same composition but different sequences of amino acids, (L -Ala-L -Val-Gly)_n and (L -Val-L -Ala-Gly)_n, have been prepared by the method involving facile monomer synthesis using N-carboxy α-amino acid anhydrides and N-hydroxysuccinimide esters. The yields and the molecular weights of the polypeptides formed by polycondensation do not depend on the monomer concentrations, but on the sequences of the amino acids in the monomers. Infrared spectra in the solid state showed that (L -Ala-L -Val-Gly)_n can take the α-helical conformation but (L -Val-L -Ala-Gly)_n cannot. The results suggest that the conformations of polypeptides are influenced by the sequences of the amino acids in the polypeptides.     

Determination of peptide conformation via vibrational coupling: Application to diastereoisomeric alanyl dipeptides

Solution-phase Raman spectra of diastereomeric alanyl dipeptides, D -Ala-L -Ala and L -Ala-L -Ala, and various mono- and dideuterated isotopomers in H₂O and D₂O, are reported. Spectral differences between the diastereomeric forms are interpreted, using the Raman analog of the coupled oscillator model, in terms of geometric differences between certain vibrations in the diastereomeric forms. Application of the coupled-oscillator formalism allows the determination of a dihedral angle between the coupling vibrations. The results are compared with vibrational coupling employed by other workers in the determination of the vibrational spectra of peptides.     

15N-nmr spectroscopy. 33. Assignments of isotactic and syndiotactic sequences in poly(D,L-amino acids)

¹⁵N-enriched (D ,L -Leu)_n, (γ-OMe-D ,L -Glu)_n, (D ,L -Val)_n, and (D ,L -Phe)_n were prepared, 40.55-MHz ¹⁵N-nmr spectra were measured in various solvents. The signal patterns depend strongly on the nature of the solvent, yet in most cases at least four signals are resolved, representing the four enantiomeric pairs of triads L -L -L (D -D -D ), L -D -L (D -L -D ), L -L -D (D -D -L ), and D -L -L (L -D -D ). Numerous copolypeptides of the general structure (A)_n-B*-(A)_m (the asterisk denotes 40–50% ¹⁵N enrichment) were synthesized and measured as models for syndiotactic sequences in the spectra of poly(D ,L -amino acids). In this way unambiguous assignments for both isotactic and syndiotactic trials were obtained. A spectroscopic rule was established: “isotactic sequences absorb downfield of syndiotactic ones.” Furthermore, the spectra of various types of stereocopolypeptides such as (L -Leu/L -Val)_n and (L -Leu/D -Val)_n were investigated, including the ternary systems (L -Leu/L -Ala/D -Ala)_n (L -Leu/L -Ala/Gly)_n, (L -Leu/D -Ala/Gly)_n, (L -Val/L -Ala/Gly)_n, and (L -Val/D -Ala/Gly)_n. All copolymerization of D - and L -amino acid NCAs investigated in this work showed a low degree of stereoselectivity.     

Experimental investigations on the backbone folding of proline-containing model tripeptides

Some proline-containing tripeptides with the general formulas R⁰CO-L -Pro-X-NHR³ (X = Gly,Sar,L -Ala,D -Ala) and R⁰CO-X-L -Pro-NHR³ (X = Gly,L -Ala,D -Ala) have been investigated in solution by ir and ¹H-nmr spectroscopies. Their favored conformational states depend mainly on both the primary structure and the chiral sequence of the molecules. In inert solvents the βII-folding mode is the most favored conformation for the L -Pro-D -Ala and L -Pro-Gly tripeptides, while the βII′-turn is largely preferred by D -Ala-L -Pro derivatives. Under the same conditions only about one-third of the whole conformers of L -Pro-L -Ala molecules adopts the βI-folding mode. Semiopened C₇C₅ and C₅C₇ conformations are appreciably populated in the L -Pro-L -Ala sequence, on the one hand, and in the Gly-L -Pro and L -Ala-L -Pro derivatives, on the other hand. In L -Pro-Sar and X-L -Pro models, the cis–trans isomerism around the middle tertiary amide function is observed. Thus cis L -Pro-Sar and L -Ala-L -Pro conformers are folded by an intramolecular i + 3 → i hydrogen bond, whereas cis D -Ala-L -Pro and Gly-L -Pro molecules accommodate an open conformation. In dimethylsulfoxide the βII- and βII′-folding modes are not essentially destabilized, as contrasted with the βI conformation, which is less populated. In water solution all the above-mentioned conformations, with the possible exception of the βII′-folding mode for D -Ala-L -Pro molecules, seem to vanish. Solute conformations are also compared with the crystal structures of four proline-containing tripeptides.     

Synthesis of poly(L-Tyr-L-Glu-L-Ala-Gly) and poly(L-Glu-L-Lys-L-Ala) and their circular dichroism spectra in aqueous solution

Two sequential polypeptides, poly(O-benzyl-L -Tyr-γ-benzyl-L -Glu-L -Ala-Gly) and poly(ε-benzyloxycarbonyl-L -Lys-L -Glu-L -Ala), were synthesized, the former by the pentachlorophenyl ester of the tetrapeptide monomer and the latter by the azide of the tripeptide monomer. After deprotection and dialysis, poly(L -Tyr-L -Glu-L -Ala-Gly) was obtained in 71% yield and had a molecular weight of 53,000. The circular dichroism spectra (CD) of the polymer at pH's 7.2, 10.5, and 11.8 and of oligomers and of the monomer at pH 7.2 indicated that the polymer exists in an α-helical conformation. After deprotection, poly(L -Lys-L -Glu-L -Ala) was obtained in 37% yield and had a molecular weight of 3000. The CD spectra of the polymer at pH 7.2 and 2.8, and of the monomer at pH 7.2, indicated that the polymer is in a randomly coiled configuration.     

The effects of isolated N-methylated residues on the conformational characteristics of polypeptides

Conformational energies have been estimated for the tripeptide fragments L -Ala-N-methyl-L -Ala-L -Ala, L -Ala-L -Ala-N-methyl-L -Ala, L -Ala-Sar-L -Ala, and L -Ala-Gly-N-methyl-L -Ala. The peptide bonds connecting L -Ala and Gly with N-methyl-L -Ala and L -Ala with Sar were permitted to adopt the planar cis as well as the usual trans conformation. Contour maps of the conformational energies of the central residue in these tripeptide fragments are presented and compared to the conformational energy maps previously calculated for unmethylated L -Ala and Gly surrounded by residues which are also unmethylated. In generl it is observed that L -Ala and Gly residues that are either N-methylated in their conformational freedom relative to the same residues in an unmethylated polypeptide chain.     

Sequential polypeptides containing arginine as histone models: synthesis and conformational studies

The sequential polypeptides (L -Arg-X-Gly)_n, where X represents amino acid residues Ala, Val, and Leu, were prepared as models of arginine-rich histones to be used in studying their structure and their interactions with DNA. The polymerization was carried out on the pentachlorophenyl active esters of the appropriate tripeptides, while the toluene-4-sulfonyl group was used for protecting the arginine guanido group. CD was employed to investigate the conformation of (L -Arg-X-Gly)_n polymers in aqueous solutions, at different pH, as well as in trifluoroenthanol and hexafluoroisopropyl alcohol solutions. In aqueous solutions (at pH 7 and 12) the prepared sequential polymers behaved as a random coil. The CD spectra in various trifluoroethanol–water or hexafluoroisopropyl alcohol–water mixtures indicated that the degree of helical conformation of the studied polytripeptides increased in the order of Ala → Val → Leu. The opposite was true for the β-structure. Characteristics of β-turn are excluded from the poly(L -Arg-L -Leu-Gly), which assumed the most pronounced helical conformation. The poly(L -Arg-L -Val-Gly) exerts a significant preference to the β-turn structure compared to that of poly(L -Arg-L -Ala-Gly). Thus the probability for helical, β-structure or β-turn conformations of the polymers was analyzed in relation to the bulkiness and length, and to the special features of the X-residue side chain (β-branching). We concluded that the prepared sequential arginine-containing polypeptides are plausible models for histone fractions, f₃ and f₂α₁.     

Raman spectroscopic study of mechanically deformed poly-L-alanine

Raman spectroscopy has been used in investigating the conformational transitions of poly-L -alanine (PLA) induced by mechanical deformation. We see evidence of the alpha-helical, antiparallel beta-sheet, and a disordered conformation in PLA. The disordered conformation has not been discussed in previous infrared and X-ray diffraction investigations and may have local order similar to the left-handed 3₁ poly glycine helix. The amide III mode in the Raman spectrum of PLA is more sensitive than the amide I and II modes to changes in secondary structure of the polypeptide chain. Several lines below 1200 cm^?1 are conformationally sensitive and may generally be useful in the analysis of Raman spectra of proteins. A line at 909 cm^?1 decreases in intensity after deformation of PLA. In general only weak scattering is observed around 900 cm^?1 in the Raman spectra of antiparallel beta-sheet polypeptides. The Raman spectra of the amide N–H deuterated PLA and poly-L -leucine (PLL) in the alpha-helical conformation and poly-L -valine (PLV) in the beta-sheet conformation are presented. Splitting is observed in the amide III mode of PLV and the components of this mode are assigned. The Raman spectrum of an alpha-helical random copolymer of L -leucine and L -glutamic acid is shown to be consistent with the spectra of other alphahelical polypeptides.     

Optical properties of polypeptides in the β-conformation

The rotational strengths and oscillator strengths of the nπ* band and ππ* exciton bands have been calculated for antiparallel and parallel β-structures of varying length and width. The results are compared with experiment and with previous theoretical treatments of β-structures. The generally good agreement of calculations on the antiparallel β-structure with experimental results on poly-L -lysine and poly-L -serine indicates that these systems are indeed in the antiparallel conformation. It is found that the exciton component strongest in absorption shifts to longer wavelengths as the width of an antiparallel structure increases, and it is suggested that the position of the ππ* absorption band may be a useful criterion of sheet width. The results also reconcile the linear dichroism measurements of Rosenheck and Sommer on poly-L -lysine films with an anti-parallel structure. Calculations on parallel β-structures indicate that the CD spectra of this form will be rather similar to that of the antiparallel form. However, the major absorption band in the antiparallel form is associated with a small positive CD band, while in the parallel form it coincides with a large negative CD band. Finally, it is pointed out that the large positive CD bands predicted for single-stranded parallel and antiparallel β-structures at about 200 mμ render unlikely the suggestion that random-coil polypeptides contain a substantial fraction of extended chain.     

Laser-Raman spectroscopy of biomolecules. XI. Conformational study of poly(L-valine) and copolymers of L-valine and L-alanine

Raman spectroscopic studies have been carried out on polymers of L -valine ranging in degree of polymerization (DP) from 2 to 930. The spectrum of the hexapeptide (DP = 6) is closely similar over the entire range 40–1750 cm^?1 to those of polymers with much higher DP, and the structure is clearly shown to be that of the antiparallel pleated sheet (β-structure) by the amide I and III frequencies. The formation of a little α-helical structure occurs in polymers with DP above 500, although the amount does not appear to be a linear function of DP. The α-helical structure is unstable and readily destroyed in samples cast from trifluoroacetic acid solution. It is stabilized by the incorporation of L -alanine, a strong helix-former; polymers of the latter may in turn be forced into a α-structure in copolymers sufficiently rich in L -valine.     

Crystal structure of polyglycine I

An electron diffraction study has been made of oriented polyglycine I (the β modification of polyglycine) and of single crystals grown from solution. The unit cell is very similar to that postulated by Astbu?y (1949). It is monoclinic with parameters a = 9.54 Å, b(chainaxis) = 7.044 Å, c = 3.67 Å and β = 113°. Examination of the possible structures suggests that polyglycine I does not have the familiar antiparallel pleated sheet, but rather the closely related antiparallel rippled sheet structure first described by Pauling &; Corey (1953a).     

Structure of apoproteins in insect lipophorin

The two polypeptide chains of cockroach and locust lipophorins were separated and their amino acid compositions were determined. Circular dichroic spectra of the lipophorins and apolipophorin from 190 to 250 nm showed a single trough at 218 nm and a peak at 194 nm. Infrared spectra of the lipophorins in D2O showed a strong peak at 1625 cm-1 and a weak shoulder at 1693 cm-1 corresponding to v (pi, 0) and nu (0, pi) of antiparallel pleated sheet. The resonance frequency splitting delta nu = nu (0, pi) -nu (pi, 0) was 68 cm-1, which was larger than that of ordinary globular proteins containing antiparallel pleated sheet. From circular dichroic and infrared spectra it was concluded that lipophorins contained polypeptides rich in antiparallel pleated sheet with longer unbroken extensions than the case for ordinary globular proteins. Partial proteolytic digestion study of lipophorins with trypsin, chymotrypsin, and subtilisin showed that the larger apolipophorin (AL1) was exposed to the surface of the particle and the smaller apolipophorin (AL2) lay protected from the attack of the enzymes. Crosslinked products between AL1 and AL2 were readily obtained when dimethylsuberimidate or dimethyladipimidate was added to the lipophorin solution, without giving lipophorin dimers, suggesting that the two chains were located within 11 A from each other. Such structural features of insect lipoprotein were compared with other insect lipophorins and the human serum low-density lipoprotein (LDL). Similarities between lipophorins and LDL were found in the molecular weight, amino acid compositions, and the secondary structure of major apoproteins.     

Synthesis of sequential oligopeptides and polypeptide having the sequence of L-alanyl-L-leucylglycine

A series of sequential oligopeptides having simple nonpolar side chains, Nps-(L -Ala-L -Leu-Gly)_n- OEt has been prepared by a stepwise fragment-condensation method using Nps-L -alanyl-L -leucylglycine N-hydroxysuccinimide ester, which was prepared by the Nps-N-carboxy α-amino-acid-anhydride method. The success of the synthesis of the peptide having a high-molecular weight, such as octadecapeptide, results from the highest solubility of the tripeptide unit, L -alanyl-L -leucylglycine. The sequential polypeptide having the same tripeptide sequence was also prepared by polycondensation of the tripeptide N-hydroxy-succinimide ester.     

Studies of peptide conformation: Backbone folding of tetrapeptide derivatives in methanol and water

Derivatives of tetrapeptide sequences considered likely to form β-turns were investigated by the study of their proton magnetic resonances in methanol and in water. Differential broadening of N—H resonances by an added nitroxyl was used to indicate the presence of the sequestered N—H proton expected in β-turn conformations. Transfer of magnetic saturation from solvent water protons to N—H protons was also examined. The evidence is consistent with significant contributions by β-turn-like backbones to the conformational averages in methanol of the sequences Gly-L -Pro-D -Val-Gly, D (or L )-Val-L -Pro-Gly-Gly, and Gly-L -Pro-L -Asn-Gly, but not the sequence Gly-D -Ala-L -Val-Gly. It is suggested that a Type I turn, Likely in Gly-L -Pro-L -Asn-Gly derivatives, is characterized by sequestered N—H protons of both the third and fourth residues. For all of the peptide derivatives, save possibly Ac-L -Val-L -Pro-Gly-Gly-NHNH₂, contributions from folded structures in water are not detectable by line-broadening experiments. However, the transfer of saturation experiments may be interpreted as indicating some degree of chain folding in water.     

Far-infrared spectra of poly(-α-amino acids) with basic alkyl group side chains

Far-infrared spectra in the region from 700 to 60 cm^?1 have been measured for the α-helix structures of poly(L -α-amino-n-butyric acid), poly-L -norvaline, poly-L -norleucine, and poly-L -leucine and for the β-form structures of poly(L -α-amino-n-butyric acid), poly-L -valine, poly(DL -amino-n-butyric acid), poly-DL -norvaline, and poly-DL -norleucine. The changes of the spectra on N-deuteration have been measured in the region between 700 and 400 cm^?1. It is concluded that, the α-helix has characteristic bauds near 690, 650, 610, 380, 150, and 100 cm^?1, and that the β-form has characteristic bands near 700, 240, and 120 cm^?1. The main-chain vibrations in the region from 600 to 200 cm^?1 are strongly coupled with the side-chain deformation vibrations.     

Structure of polyglycine I: a comparison of the antiparallel pleated and antiparallel rippled sheets

Packing energy calculations are made for two possible sheet structures of polyglycine I, i.e. the antiparallel pleated and rippled sheets. They indicate that the rippled sheet is the more stable structure and that its calculated lattice parameters are close to those experimentally determined. Furthermore, the results on the packing of pleated sheets of polyglycine improve understanding of the well-known model of silk fibroin structure of Marsh et al. (1955). They also suggest that the sheet structures of l-polypeptides with short side-chains should pack in monoclinic unit cells rather than the orthorhombic ones which are observed. A possible origin of this discrepancy is discussed.     

A crystal structure with features of an antiparallel α-pleated sheet

A single-crystal x-ray diffraction analysis of Boc-L -Ala-D -aIle-L -Ile-OMe has been carried out. The analysis has shown (a) that the tripeptide molecules have in part an α-extended conformation, the torsion angles of the L -Ala and D -aIle residues being φ₁ = ?75.1° and ψ₁ = ?25.8° and φ₂ = 67.3° and ψ₂ = 44.1°, respectively, and (b) that the molecules are organized in rippled planes where they occur in relative antiparallel orientation linked together side by side by H bonds. This molecular organization of the tripeptide corresponds closely to that of an antiparallel α-pleated sheet, and likely constitutes the first example of a structure of this kind for which a characterization at the atomic level has been achieved. A molecular dynamics study has shown that the molecular conformation of the tripeptide in the crystalline state is determined primarily by intermolecular interactions. © 1994 John Wiley & Sons, Inc.     

Secondary structures of peptides: 5 · 15N n.m.r. CP/MAS spectroscopy of solid polypeptides and gramicidin-S

30.5 MHz ¹⁵N m.m.r. (CP/MAS) spectra of various solid polypeptides were measured using the cross-polarization/magic angle spinning technique. In order to obtain optimum signal-to-noise ratios, relatively short contact times (1 ± 0.5 ms) are required, because the cross-polarization times (T_NH) are short and because the proton rotating-frame relaxation times (T_1p) are in the order of 20 ms. The ¹⁵N n.m.r. signals of copolypeptides may be sensitive to sequence effects; yet they are in most cases more sensitive to the nature of the secondary structure. The signals of α-helices absorb ca. 8–10 ppm upfield of β-sheet structures, whereas the polyglycine II helix absorbs downfield. The natural abundance spectrum of crystalline gramicidin-S exhibits a signal at ?247 ppm, a characteristic chemical shift of the antiparallel pleated sheet structure.     

Conformational analysis of the beta sheet structure of poly-L-alanine and poly(L-alanine-glycine).

The β pleated sheet structures of poly-l-alanine and the polydipeptide (Ala-Gly)_n are analysed by conformational energy calculations, and the results are compared with the structures previously determined by X-ray methods. Structural parameters calculated for β poly-l-alanine using two different sets of potentials are in close agreement (within 5% or less) with the results of X-ray structure analysis (Arnott et al., 1967). For (Ala-Gly)_n, the alanyl and glycyl-glycyl intersheet distances calculated by assuming the model of Fraser et al. (1965) are comparable to those observed in the polydipeptide, but differ significantly from those of (Gly)_n and (Ala)_n. These calculations help establish the structural details of both the polydipeptide and the closely related Bombyx mori silk protein in their β form.     

Lysine oligopeptides. Preparation by ion-exchange chromatography

The preparation of L -lysine peptides (Lys_n, n = 2–14) from polyL -lysine is described. Fractionation by ion-exchange column chromatography of poly-L -lysine hydrolysates on a preparative scale resulted in 0.2–1.0 g quantities of individual members of the poly-L -lysine series. The peptides isolated proved to be analytically pure and the optical configuration was fully retained, as demonstrated by complete enzymic digestion. Peptides higher than n = 14 were also prepared. They consisted of oligolysine groups of narrow and accurately determined size distribution. Potentiometric titrations were used both to characterize the products and to demonstrate the characteristic dependence of the dissociation constants on size of the peptide.