Vibrational CD studies of the solution conformation of simple alanyl peptides as a function of pH

The CH-stretching vibrational CD (VCD) spectra of glycyl-L-alanine, L-alanylglycine, and L-alanyl-L-alanine have been studied at neutral, high, and low pH in D2O solution. The intense positive VCD band attributed to the C alpha H stretch of the alanyl residue in glycyl-L-alanine at neutral pH is absent in L-alanylglycine. In contrast to the VCD spectra of L-alanine, the positive methine-stretching VCD band in glycyl-L-alanine and L-alanyl-L-alanine is still present at pH 2. Based on the ring current mechanism, the VCD spectra are consistent with the presence of a five-membered CO...HN intramolecular hydrogen-bonded ring between the C-terminal carboxylate and peptide NH groups at neutral and high pH; and a seven-membered COH...O = C hydrogen-bonded ring between the C-terminal carboxyl OH and peptide C = O groups at low pH. In the N-terminal alanyl residue, the peptide C = O group is hydrogen bonded to the NH trans to the methine bond. The CH-stretching VCD spectra of L-alanyl-L-alanyl-L-alanine at neutral pH are consistent with two intramolecularly hydrogen-bonded conformations for the central alanyl residue.     

Synthesis and solution conformation studies of 3-substituted uridine and pseudouridine derivatives

A series of 3-substituted uridine and pseudouridine derivatives, based on the naturally occurring 3-(3-amino-3-carboxypropyl) modification, were synthesized. Their aqueous solution conformations were determined by using circular dichroism and NMR spectroscopy. Functional group composition and chain length were shown to have only a subtle influence on the distribution of syn/anti conformations of the modified nucleosides. The dominating factor appears to be the glycosidic linkage (C- vs. N-glycoside) in determining the nucleoside conformation.     

The conformation of single-strand polynucleotides in solution: sedimentation studies of apurinic acid

To elucidate the role of the bases in single-strand polynucleolide conformation, we have studied apurinic acid, a single-strand polydeoxyribonucleotide from which almost half the bases have been removed. The conformation of apurinic acid in aqueous solution near θ condition has been investigated by sedimentation velocity and sedimentation equilibrium measurements. The unperturbed coil dimensions of apurinic acid are essentially identical to those of poly rU of the same degree of polymerization. The dimensions are also similar to those of poly rA at high temperature, where the adenine residues are not stacked upon one another. We conclude that the considerable rigidity of these polynuclotides is conferred not by residual, undetected base stacking, but by restrictions in rotation about the bonds of the backbone. Furthermore, the rigidity of the ribose-phosphate backbone cannot be attributed to interactions involving the 2'—OH group.     

Vibrational CD studies of interchain hydrogen-bonded tripodal peptides.

The solution conformations of three trispeptides--L,L,L-1,3,5-C6H3[CH2NHCOCH(X)-NHBoc++ +]3, X = CH3 (Ala) or CH2CH(CH3)2 (Leu), and L,L,L-N(CH2CH2NHCOCH[CH2-CH(CH3)2]NHBoc)3--have been determined from their ir and vibrational CD (VCD) spectra in the NH stretching and carbonyl stretching regions in apolar solution. The compounds containing L-Leu are shown to occur primarily in a propeller conformation with C3 symmetry that is stabilized by interchain hydrogen bonds. Through application of the coupled oscillator model of VCD, a right-handed sense for the hydrogen-bonded chains in the propeller is deduced, in agreement with previous empirical force field calculations. The spectra also provide evidence for interchain association between two chains, resulting in a C10-ring. For chains not involved in interchain association, the spectra reveal the presence of C7-rings within a chain. The trispeptide containing L-Ala is found to occur primarily in a random form.     

Fluorescence and CD studies on the conformation of the gastrin releasing peptide in solution and in the presence of model membranes

The conformation of the heptacosapeptide hormone, gastrin releasing peptide, has been studied in buffer and in the presence of lipids, using static and dynamic fluorescence and CD. The results obtained show that, in buffer, the hormone exists in a collection of flexible, random coil type conformers, characterized by a beta-turn between residues 14-19. On the other hand, organic solvents can induce some degree of ordered secondary structure in the peptide chain. The marked changes, observed in CD and fluorescence spectra upon addition of lysolecitin micelles and dimyristoylphosphatidylserine vesicles, clearly show that the peptide interacts with lipids, assuming a lipid specific configuration. Interestingly, no significative spectroscopic changes are produced by exposure to dimyristoylphosphatidylcholine vesicles both in the gel and liquid-chrystalline phases, suggesting a requirement for negatively charged lipids during the process of hormone-membrane interaction.     

Nuclear magnetic resonance studies of histone IV solution conformation.

The 220-MHz high-resolution proton magnetic resonance (PMR) spectrum of histone IV has been examined as a function of histone concentration, salt concentration, and pD. The hydrophobic C-terminal portion of the histone IV monomer appears to be largely PMR "invisible" indicating that this region of the polypeptide contains rigid secondary structure. Further loss of PMR resonance areas with increased histone IV concentration in neat D2O has been attributed to self-aggregation involving a monomer-dimer equilibrium. An equilibrium between the monomer and large aggregates, on the other hand, appears to dominate at NaCl concentrations above 0.01 M. pD studies reveal an abrupt increase in histone IV aggregation at pD smaller than 0.8 and precipitation of histone IV at pD values in the neighborhood of its isoelectric point, pD similar to 11.     

NMR studies of the solution conformation of the sex peptide from Drosophila melanogaster

The insect sex peptide (SP) elicits a variety of biological responses upon transfer to the mated female. SP contains 36 amino acids, including a tryptophan-rich N-terminal region, a central region containing five hydroxyproline (Hyp) residues, and a C-terminal region enclosed by a disulfide bridge. The solution structure of SP, studied here using NMR spectroscopy, includes a motif WPWN that adopts a type I β-turn in the N-terminal Trp-rich region. This turn region is connected to the central Hyp-rich region, which adopts extended and/or PPII-like conformations. The C-terminal disulfide-bonded loop populates helical turns or nascent helical structure. Overall, the results reveal a rather flexible peptide that lacks a compact folded structure in solution.     

Vibrational Raman optical activity of alanyl peptide oligomers: A new perspective on aqueous solution conformation

The vibrational Raman optical activity (ROA) spectra of di- and tri-L -alanine in the range 650–1750 cm^?1 have been measured in H₂O and D₂O solution at high, neutral, and low pH and pD. Corresponding ROA spectra for tetra- and penta-L -alanine have also been obtained, but over a more restricted set of pH and pD conditions. There are similarities with the ROA spectrum of L -alanine below ～ 1200 cm^?1, but the spectra are very different above this wavenumber due to the influence of the vibrational coordinates of the peptide group. The similar overall appearance of the di-, tri-, and tetrapeptide ROA under selected conditions of pH and pD, and of all four peptide ROA spectra in DCl and HCl solutions, in the backbone skeletal stretch region ～ 1050–1200 cm^?1 and the extended amide III region ～ 1250–1350 cm^?1, suggests that the backbone conformation is approximately the same in all four structures. One difference, however, is a shift of a large positive ROA band in H₂O at ～ 1341 cm^?1 in the dipeptide, assigned to C_α–H and in-plane N–H deformations, down to ～ 1331 cm ^?1 in the tripeptide and to ～ 1315 cm^?1 in the tetrapeptide and pentapeptide (the last in HCl due to insufficient solubility in H₂O), which indicates increasing delocalization of the corresponding normal mode with increasing chain length. Our results do not support the suggestion that stabilizing interactions of the zwitterionic end groups in tri-L -alanine at neutral pH leads to a different solution structure to that at high pH. © 1994 John Wiley & Sons, Inc.     

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.     

Adenine nucleosides in solution: circular dichroism studies and base conformation.

Adenosine, AMP, S-adenosylhomocysteine, S-adenosylmethionine, aristeromycin and 25 other synthetic adenosine analogs modified in the 4' or 5' positions show certain groups of different circular dichroism (CD) spectra. Both positive and negative Cotton effects can occur in the long-wavelength part (250-270 nm) of the spectra. Molar ellipticities [theta] range from -6000 (in adenosine 5'-carboxylate) to +4000 deg. cm2 dmol-1 (in 5'-deoxy-5'iodoadenosine), including some compounds with small, polar 5'-substituents in which low-intensity bands are found in signed pairs. Most of these adenosine derivatives that have the same adenine chromophore and a ribofuranose moiety unsubstituted in the 2' and 3' positions prefer an anti-conformation of the adenine base, as evidenced by proton magnetic resonance spectroscopy. In the majority of cases, electronic perturbations of the chromophore or major alterations of the assymmetric sugar residue can be excluded as sources of the CD variations. Therefore a correlation of the long-wavelength CD bands with the glycosyl torsion angle phiCN is suggested, where the gauche, gauche/anti combination which is typical of AMP in the crystal and in solution (phiCN approximately -40degrees, [theta] negative) is one reference point and a region for phiCN = 0degrees ([theta] positive) is assigned to compounds with space-filling substituents such as S-adenosylmethionine. Both negative and positive Cotton effects can be associated with the anti conformation range. Within this series, the base conformation of novel nucleoside structures could be predicted from CD measurements. The CD spectrum gives no indication, however, of whether a certain torsion angle is the result of a rigid structure (as in AMP) or the average value of a molecule with high rotational freedom (as in 5'-deoxyadenosine). The conformations of aristeromycin and 4'-thioadenosine are discussed in relation to adenosine, and a structure-determining effect of the 4' bridge atom is noted.     

Nuclear magnetic resonance studies of the solution conformation of nucleoside diphosphohexoses and their components.

The solution conformations of UDPG, UDPGN, UDPGal, UDPM, UDPGluc, UDPGalc, ADPG, ADPM, GDPG, GDPM, and CDPG and their components Glu-1-P, Gal-1-P, Man-1-P, Gluc-1-P, Galc-1-P, ADP, GDP, UDP, and CDP are studied by high resolution fast Fourier transform nuclear magnetic resonance spectroscopy with iterative computer line shape simulation. The following results were observed. (1) The six-membered ring is in 4C1 chair form with the C(5')-C(6') bond in gg equilibrium tg equilibrium for the derivatives of glucose and mannose and gt equilibrium tg for those of galactose. (2) No conformational preference can be detected for C(1')-O(1') bond in hexose-1'-P moiety. (3) Chemical shift dependencies for the pyranoid ring protons and their structural and conformational relations are: (a) axial proton is at higher field than equatorial: (b) the shielding effect of a gauche vicinal hydroxyl group is stronger than a trans vicinal; (c) the vicinity of a hydroxyl group located more than three bonds away tends to shift the proton downfield. (4) The conformation of the nucleoside 5'-diphosphate part is [anti, 2E equilibrium 3E, g'g' equilibrium g't', g'g' equilibrium g'/t'], with slight variation of each conformation occuring for individual compounds. (5) No significant interactions are detected between the hexose and nucleoside parts in the nucleoside diphosphohexoses, and the hexose and nucleoside components display the same conformational preference as they become integrated to form nucleoside diphosphohexoses.     

Proton NMR and CD solution conformation determination and opioid receptor binding studies of a dynorphin A(1-17) model peptide

The opioid peptide dynorphin A(1-17) contains a peptide segment in residues 7-15 with the potential to form an amphiphilic beta-strand. This amphiphilic structure may, like the amphiphilic alpha-helices found in many other peptide hormones, be an important determinant of its interactions with membranes and receptors. In order to investigate and characterize these interactions, we have synthesized a 17-residue dynorphin analogue (YGGFLKKVKPKVKVKSS) that incorporates a peptide model of this amphiphilic secondary structure with minimized homology (25%) relative to the native sequence. This peptide exhibits the full biological potency of dynorphin in assays of kappa-opioid receptor binding, and is more selective for this type of opioid receptor than the natural peptide. The conformation of the model peptide in aqueous solution has been investigated in detail by NMR spectroscopy. The values of the NH-CH alpha coupling constants together with rotating frame NOEs indicate the presence of an amphiphilic structure together with some beta-strand structure in residues 7-15, and demonstrate that a peptide model that stabilizes this structure in aqueous solution and enhances kappa-opioid receptor selectivity can be successfully designed using using alternating lysine and valine residues.     

Coupling of iminodiacetic acid with amino acid derivatives in solution and solid phase

Summary The IR studies for the preactivation step of N-protected iminodiacetic acid with different coupling reagents (TCFH, TFFH, HATU, HBTU, HSTU) were reported here and showed the formation of an anhydride as an active intermediate in case of TCFH and TFFH. The formation of a mixture of an anhydride and an active ester (-OBt,-OAt or-OSu) were observed for HBTU, HATU or HSTU coupling reagent. Dependent on the coupling conditions, acylation of N-protected iminodiacetic acid with amino acid ester or amide derivatives in solution phase gave monoor di-substituted iminodiacetic acid derivatives. Coupling of N-protected iminodiacetic acid with an amino acid or peptide attached to a solid support (PAL-PEG-PS or Wang resin) gave only the monosubstituted iminodiacetic acid derivatives. Abbreviations: HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; Boc,t-butyloxycarbonyl; DCC, N,N′-dicyclohexylcarbodiimide; DIC, N,N′-diisopropylcarbodiimide; DIEA, diisopropylethylamine; HATU, N-[(dimethylamino)-1H-1,2,3,-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; DMF, N,N-dimethylformamide; Bsmoc, 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl; Fmoc, 9-fluorenylmethyloxycarbonyl; HOAt, l-hydroxy-7-azabenzotriazole; HOBt, l-hydroxybenzotriazol; IDA, iminodiacetic acid; HSTU, O-(succinimidyl)-tetramethyluronium hexafluorophosphate; TCFH; 1,1,3,3-tetramethyl-2-chloroformamidinium hexafluorophosphate; TFFH, 1,1,3,3-tetramethyl-2-fluoroformamidinium hexafluorophosphate; TMS-Cl, trimethylchlorosilane. Amino acids and peptides are abbreviated and designated following the rules of the IUPAC-IUB Commission of Biochemical Nomenclature (J. Biol. Chem., 247 (1972) 997).     

Coupling of iminodiacetic acid with amino acid derivatives in solution and solid phase

The IR studies for the preactivation step of N-protected iminodiacetic acid with different coupling reagents (TCFH,TFFH, HATU, HBTU, HSTU) were reported here and showed theformation of an anhydride as an active intermediate in caseof TCFH and TFFH. The formation of a mixture of an anhydrideand an active ester (–OBt, –OAt or –OSu) were observed forHBTU, HATU or HSTU coupling reagent. Dependent on the couplingconditions, acylation of N-protected iminodiacetic acid with amino acid ester or amide derivatives in solution phase gavemono- or di-substituted iminodiacetic acid derivatives. Couplingof N-protected iminodiacetic acid with an amino acid or peptideattached to a solid support (PAL-PEG-PS or Wang resin) gave onlythe monosubstituted iminodiacetic acid derivatives.     

X-ray diffraction studies of the conformation of macromolecules in solution. 3. The helix-coil transition

The procedure outlined in the previous paper of this series is applied to x-ray diffraction patterns of labeled polytyrosine in dimethylformamide–trifluoroacetic acid solutions. The α-helix appears to transform into the random coil conformation at a trifluoroacetic acid concentration between 40 and 42.5%. Comparison of these results with those measured in the same system by optical rotation are in essential agreement.