The Ac-RGD-NH2 peptide as a probe of slow conformational exchange of short linear peptides in DMSO

According to general belief, the conformational information on short linear peptides in solution derived at ambient temperature from NMR spectrometry represents a population-weighted average over all members of an ensemble of rapidly interconverting conformations. Usually the search for discrete conformations is concentrated at low temperatures especially when sharp NMR resonances are detected at room temperature. Using the peptide Ac-RGD-NH(2) (Ac-Arg-Gly-Asp-NH(2), Ac: acetyl) as a model system and following a new approach, we have been able to demonstrate that short linear peptides can adopt discrete conformational states in DMSO-d(6) (DMSO: dimethylsulfoxide) which vary in a way critically dependent on the reconstitution conditions used before their dissolution in DMSO-d(6). The conformers are stabilized by intramolecular hydrogen bonds, which persist at high temperatures and undergo a very slow exchange with their extended structures in the NMR chemical shift time scale. The reported findings provide clear evidence for the occurrence of solvent-induced conformational exchange and point to DMSO as a valuable medium for folding studies of short linear peptides.     

The conformational restriction of synthetic peptides, including a malaria peptide, for use as immunogens

A new strategy is advanced for the conformational restriction of peptidyl immunogens. Our approach is to replace putative amide-amide hydrogen bonds with covalent hydrogen-bond mimics. Because on average every other amino acid in a protein engages in this bond, the syntheses of diversely shaped peptides can be contemplated. Synthetic methods for introducing a potential hydrogen-bond mimic into a peptide with alpha-helical potential is reported and the structural consequences are discussed. The replacement of the hydrogen bond with a chemical link will modify as well as shape the peptide. To explore the consequences of these changes, a potential synthetic vaccine for malaria, the repeating tetrapeptide Asn-Pro-Asn-Ala, was conformationally restricted. Antibodies to the shaped malarial peptide showed a strong cross reaction with Plasmodium falciparum sporozoites.     

Synthesis and nmr conformational studies of polyoxyethylene-bound, alpha-deuterated glutamate oligopeptides

The syntheses of three series of glutamate oligopeptides attached to a macromolecular solubilizing polyoxyethylene (POE) group Boc-[Glu(OMe)]_n-OPOE, Ac-[Glu(OMe)]_n-OPOE, pGlu-[Glu(OMe)]_n?1-OPOE (n ? 1–7) and their various analogs specifically deuterated at individual α-CH positions using the liquid-phase method of peptide synthesis are described. It was shown that stepwise synthesis using the symmetrical anhydride gave homo-oligopeptides that are analytically pure. Fragment condensation methods using DCC-HOBt yield POE-peptides with POE-HOBt impurities but the peptide synthesis may be carried stoichiometrically with smaller quantities of amino acid derivatives. 360 MHz ¹H-nmr conformational studies of these homo-oligopeptides in DMSO-d₆ are presented. The α-deuterated peptides are shown to allow unequivocal homoligopeptide backbone NH assignments.     

A simple hydrogen bond potential function for conformational studies

Since hydrogen bonding plays an important role in determining energetically favourable conformations of biological molecules, a computationally simple and convenient semiempirical function has been evaluated by constraining a function to conform to the experimentally observed data for isolated simple systems of hydrogen bonded dimers. The function is found to be satisfactory for use in conformational energy calculations.     

Hydrogen-1 and carbon-13 nuclear magnetic resonance conformational studies of the His-Pro peptide bond: conformational behavior of TRH

Cis/trans isomerism of the His-Pro peptide bond provides a convenient model for the effect of a slow conformational change which may have wider biological significance. Above the imidazole pK, His-Pro is conformationally analogous to the (isosteric) peptide Phe-Pro. Protonation of the imidazole sidechain is associated with a large decrease in the cis/trans ratio. Detailed 1H and 13C n.m.r. analysis suggests the importance of electrostatic/hydrogen bonding interactions between the charged imidazolium sidechain and the proline carboxyl as the basis for this effect. In contrast to a previous report, cis/trans isomerism in TRH is shown to be related to titration of the imidazole sidechain, exhibiting a pK of 6.1.     

Synthesis and conformational analysis of N-glycopeptides. II. CD,molecular dynamics,and nmr spectroscopic studies on linear N-glycopeptides

The comprehensive structural analysis reported herein of eight N-glycopeptides, in three different solvents, is based on quantitative CD experiments, homonuclear nuclear Overhauser effect measurements, and molecular dynamics (MD) calculations. Although several orientations of the two amide planes attached to the carbohydrate pyranose ring are possible, according to NOE, CD data, and MD simulations, of all of the glycopeptide models, regardless of the type of the carrier peptide, only one dominant conformer population was found. This conformer is characterized by a nearly trans orientation of the CH and NH hydrogens of both acetamido groups. This finding is in perfect agreement with x-ray crystallographic data on the solid state conformation of the 1-N-acetyl- and 1-N-(β-aspartoyl)-2-acetamido-2-deoxy-β-D -glucopyranosylamine. The precise identification of this dominant conformer of N-glycopeptides in solution was the major question addressed herein by the structural analyses. A “CD additivity” experiment was carried out using an equimolar solution of Boc-Pro-Asp-NHCH₃ and l-N-acetyl-3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D -gluco-pyranosylamine at ambient temperature in acetonitrile. The CD spectrum obtained from the equimolar solution of the above two molecules (the “spectroscopic sum”) was identical with the CD curve obtained from the algebraic summation of the individually recorded CD spectra of the peptide and the carbohydrate moiety (“mathematical sum”). The global picture of the CD spectral analyses of the eight parent peptides with the eight N-glycopeptides revealed that in trifluoroethanol and acetonitrile, the side-chain modification of the Asn models (natural N-glycopeptide analogues) by N-glycosylation has a significant effect on the conformation of the carrier peptide, resulting in a decrease in the original type I β-turn content. Simultaneously, the type II β-turn conformational percentage increased to ≈ 20%. Such a conformational ratio change seems to be larger than the expected errors arising from the CD analyses, and agrees with the results of MD calculations. N-glycosylation of Asn residues causes perturbations, not only through the covalent bond, but also through specific hydrogen bonds between the backbone and side chain atoms. CD spectroscopy, augmented by efficient CD curve deconvolution techniques, has proved to be a useful tool for studying multicomponent conformer mixtures of small linear peptides in solution and changes of conformational equilibria caused by N-glycosylation. © 1993 John Wiley & Sons, Inc.     

Conformations of dehydrophenylalanine containing peptides: nmr studies of an acyclic hexapeptide with two delta Z-Phe residues

The conformation of an acyclic dehydrophenylalanine (delta Z-Phe) containing hexapeptide, Boc-Phe-delta Z-Phe-Val-Phe-delta Z-Phe-Val-OMe, has been investigated in CDCl3 and (CD3)2SO by 270-MHz 1H-nmr. Studies of NH group solvent accessibility and observation of interresidue nuclear Overhauser effects (NOEs) suggest a significant solvent-dependent conformational variability. In CDCl3, a population of folded helical conformations is supported by the inaccessibility to solvent of the NH groups of residues 3-6 and the detection of several NiH----Ni + 1H NOEs. Evidence is also obtained for conformational heterogeneity from the detection of some Ci alpha H----Ni + 1H NOEs characteristic of extended strands. In (CD3)2SO, the peptide largely favors an extended conformation, characterized by five solvent-exposed NH groups and successive Ci alpha H----Ni + 1H NOEs for the L-residues and Ci beta H----Ni + 1H NOEs for the delta Z-Phe residues. The results suggest that delta Z-Phe residues do not provide compelling conformational constraints.     

Heme as an optical probe of a conformational transition of ovine recPrP

The prion protein occurs as a globular domain and a leading fragment whose structure is not well-defined. For the ovine species, all of the tryptophan residues are in the initial fragment, while the globular domain is rich in tyrosine residues. Using heme as a spectroscopic probe, we have studied the recombinant prion protein before and after a temperature-induced conformational change. As for most heme proteins, the absorption spectrum of heme-CO displays a red shift upon binding to the protein, and both the Y and W fluorescence are highly quenched. Flash photolysis kinetics of the PrP-heme-CO complex shows a low yield for the bimolecular phase, indicating a pocket around the hemes. By comparing the holoprotein and the truncated sequence corresponding to the globular domain, the stoichiometry was determined to be five hemes for the globular domain and two hemes for the leading fragment. At high temperature, the hemes are released; upon cooling, only two hemes bind, and only the tryptophan fluorescence is quenched; this would indicate that the globular domain has formed a more compact structure, which is inert with respect to the hydrophobic probe. The final state of polymerization is perturbed if the synthetic peptide "N3" (PrP residues 142-166, which include the first helix) is added to the prion protein solution; the temperature cycle no longer reduces the number of heme binding sites. This would indicate that the peptide may alter or inhibit the polymer formation.     

Structural features of linear (alphaMe)Val-based peptides in solution by photophysical and theoretical conformational studies

In continuation of our studies on the determination of the structural features of functionalized peptides in solution by combining time-resolved fluorescence data and molecular mechanics results, the conformational features of a series of linear, L-(alphaMe)Val-based peptides have been investigated in methanol. These foldamers have the general formula F[(alphaMe)Val](r)-T-[(alphaMe)Val](2)NHtBu, where (alphaMe)Val = C(alpha)-methylvaline and r = 0-3, while F [= fluoren-9-ylmethoxycarbonyl (Fmoc)] and T [= 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-carboxylic (Toac)] are a fluorophoric N(alpha)-protecting group and a nitroxide-based alpha-amino acid quencher, respectively. According to ir and CD spectra, the longest term of the series (r = 3) attains a 3(10)-helical structure, while the other peptides populate an intramolecularly H-bonded, 3(10)-helix-like conformation affected by dynamic helical distortions, which are enhanced by the shortness of the backbone chain. Such distortions are reflected in both the energy of the stretching mode and the molar extinction coefficient of the H-bonded N-H groups, the former being higher and the latter smaller than those of a stable 3(10)-helix. Steady-state and time-resolved fluorescence measurements in methanol show a strong quenching of Fmoc by the Toac residue, located at different helix positions, depending on the r value. Comparison of quenching efficiencies and lifetime preexponents with those theoretically obtained from the deepest energy minimum conformers, assuming a F?rster mechanism, is satisfactory. The computed structures exhibit a rather compact arrangement, which accounts for the few sterically favored conformations for each peptide, in full agreement with the time-resolved fluorescence data. Orientational effects between the probes must be taken into account for a correct interpretation of the fluorescence decay results, implying that interconversion among conformational substates involving the probes is slower than the energy transfer rate.     

Fourier transform vibrational circular dichroism as a decisive tool for conformational studies of peptides containing tyrosyl residues

Previous UV-circular dichroism (UV-CD) and NMR studies showed that Ac-AAAAAAAEAAKA-NH(2) has an alpha-helical structure in 50% (v/v) aqueous trifluoroethanol. Replacement of Ala(1) to Ala(6) with Tyr results in spectra that show an apparent loss of helicity in the same solvent. This apparent loss of helicity could be attributed to the coupling of the tyrosyl side chain chromophore with the backbone amide. However, such electronic coupling does not affect the vibrational CD (VCD) spectra. The VCD spectra of the peptides with tyrosyl residues were identical to that of the peptide containing no Tyr, which shows the same alpha-helical structure. Because it is now clear that Tyr replacement does not change the backbone conformation of peptides, UV-CD measurements should be complemented by VCD to determine the secondary structure when electronic effects can disturb the UV-CD spectrum of the inherent structure.     

Tryptophan as a probe for acid-base equilibria in peptides

We present results of time resolved fluorescence measurements performed in Tryptophan (Trp) derivatives and Trp-containing peptides in the pH range 3.0-11.0. For each compound a set of decay profiles measured in a given range of pH values was examined as a whole, using the global analysis technique. The data were fitted to two or three lifetime components and the analysis allowed the monitoring of the changes in the concentration of the different species contributing to the total fluorescence in that pH interval. The decay components were sensitive to the ionization state of groups neighboring the indol ring, and pK values for the equilibrium between protonated and deprotonated species were obtained from the preexponential factor of the lifetime components. In Trp, protonation of the amino terminal of the rotamer having electron transfer rate comparable to fluorescence decay rates was responsible for the interconvertion of a long lifetime component, to the 2.9 ns component usually observed in neutral pH. Trpbond;X peptides also have a single rotamer dominating the decay that is quenched by NH(3) (+). X-Trp peptides seem to be conformationally less restricted, and it is possible that rotamers interconvertion occur in high pH, increasing the population of nonquenched rotamers. Interconvertion between rotameric conformations of Trp are also present in the titration of ionizable groups in the side chain of peptides like His-Trp and Glu-Trp and control of pH is essential to the correct interpretation of fluorescence data in the study of peptides having such groups near to the Trp residue.     

The binding of 2,3-diphosphoglycerate as a conformational probe of human hemoglobins

Since 2,3-diphosphoglyeerate preferentially binds to deoxygenated hemoglobin A, this binding reaction can be used to detect the change in quaternary conformation of hemoglobin associated with the change in ligand state of the hemes. We have studied the binding to two M hemoglobins (M_HydePark, M_Milwaukee-1) that have the substituted chains in the ferric state, as well as to the mixed liganded hybrids α1₂β² and α²β1₂ (1 heme in cyanmet form) prepared from hemoglobins A and H. The studies demonstrate that when these hemoglobin variants and derivatives are deoxygenated, they bind the organic phosphate to an extent similar, but not identical, to that for fully deoxygenated hemoglobin A. The results indicate that removal of ligand from only two of the four hemes results in a change in quaternary structure to a deoxy-like conformation.     

Strategies for dealing with conformational sampling in structural calculations of flexible or kinked transmembrane peptides.

Peptides corresponding to transmembrane (TM) segments from membrane proteins provide a potential route for the determination of membrane protein structure. We have determined that 2 functionally critical TM segments from the mammalian Na+/H+ exchanger display well converged structure in regions separated by break points. The flexibility of these break points results in conformational sampling in solution. A brief review of available NMR structures of helical membrane proteins demonstrates that there are a number of published structures showing similar properties. Such flexibility is likely indicative of kinks in the full-length protein. This minireview focuses on methods and protocols for NMR structure calculation and analysis of peptide structures under conditions of conformational sampling. The methods outlined allow the identification and analysis of structured peptides containing break points owing to conformational sampling and the differentiation between oligomerization and ensemble-averaged observation of multiple peptide conformations.     

Mass spectrometric and chemical stability of the Asp-Pro bond in herpes simplex virus epitope peptides compared with X-Pro bonds of related sequences.

The mass spectrometric analysis of the immunodominant epitope region (273-284) of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) showed a favoured fission at the Asp-Pro peptide bond. The fast atom bombardment collision induced dissociation (FAB-CID) study of closely related X-Pro peptides documented that neither the length nor the amino acid composition of the peptide has a significant influence on this preferential cleavage. At the same time the DP bond proved to be sensitive to acidic conditions in the course of peptide synthesis. These observations prompted us to compare the chemical and mass spectrometric stability of a new set of nonapeptides related to the 273-284 epitope region of gD, i.e. SALLEDPVG and SALLEXPVG peptides, where X = A, K, I, S, F, E or D, respectively. The chemical stability of these peptides during acidic hydrolysis was investigated by electrospray ionization mass spectrometry (ESI-MS) and the products were identified by ESI-MS and on-line high performance liquid chromatography-mass spectrometry (HPLC-MS). The mass spectrometric fragmentation and bond stability of the untreated peptide samples were also studied using ESI-MS and liquid secondary ion mass spectrometry (LSIMS). Both the chemical hydrolysis and the mass spectrometric fragmentation showed that the Asp-Pro bond could easily be cleaved, while the KP bond proved to be stable under both circumstances. On the other hand, the XP bond (X = A, I, S, F or E) fragmented easily under the mass spectrometric conditions, but was not sensitive to the acidolysis.     

Amination of carboxyl-terminal homoserine peptides as an aid in peptide separation.

Lactonization and amination of peptide mixtures containing C-terminal homoserine peptides facilitated separation of these peptide mixtures. The use of radio-actively labeled diamine allowed easy identification of the carboxyl terminal peptide in a cyanogen bromide generated digest. Ambiguities arising from mixtures of homoserine and homoserine lactone forms of peptides were resolved following amination of these mixtures. A C-terminal homoserine peptide was selectively removed from a mixture of nonhomoserine peptides.     

Improving efficiency of an angiotensin converting enzyme inhibitory peptide as multifunctional peptides

Bioactive peptides have been defined as specific protein fragments that have numerous biological activities. The aim of this study was to introduce three multifunctional peptides. Hence, we used rabbit lung angiotensin converting enzyme (ACE) inhibitor peptide AFKDEDTEEVPFR to prepare two analogous peptides KDEDTEEVP and KDEDTEEVH. ACE inhibitory, antioxidant, and antimicrobial activities of three synthetic peptides were investigated. Among the three peptides, KDEDTEEVP exhibited the highest ACE inhibitory activity with IC₅₀ value of 69.63 ± 2.51 μM. Furthermore, the results of fluorescence spectroscopy and molecular modeling showed that KDEDTEEVP had more affinity to ACE than other peptides. The peptide of KDEDTEEVH showed the strongest antioxidant scavenging capacity on DPPH radicals (EC₅₀ = 135 ± 9.62 μM), hydroxyl radicals (EC₅₀ = 144 ± 8.73 μM), and ABTS radicals (EC₅₀ = 62 ± 4.52%). Moreover, it showed the highest activity in iron-chelating test (EC₅₀ = 226 ± 14.13 μM) and could also effectively inhibit the peroxidation of linoleic acid. The antimicrobial activity results showed that KDEDTEEVH had higher efficiency against Gram-positive than Gram-negative bacteria with MIC values of higher than 205 ± 10.75 μM. Although there was not a direct correlation between ACE inhibitor and antioxidant activity for analogous peptides, both analogous peptides exhibited more efficiency than the mother peptide. Thus, they can be considered as multifunctional peptides and would be beneficial ingredient to be used in food and drug industry.     

Synthesis and conformational study of two L-prolyl-L-leucyl-glycinamide analogues with a reduced peptide bond

The peptide bond between Pro-Leu or Leu-Gly in Pro-Leu-Gly-NH2 was replaced by a CH2-NH function. The 1H and 13C n.m.r. studies demonstrated that HCl X Pro-Leu psi (CH2-NH)Gly-NH2 10 adopted a conformation in DMSO that is similar to the previously postulated beta-turn for the natural hormone. This was not the case for the other analogue. In vivo tests on 10 revealed an activity approximately equal to the natural compound and an increased toxicity.     

Synthetic peptides as probes for conformational preferences of domains of membrane receptors

Peptide models have been widely used to investigate conformational aspects of domains of proteins since the early 1950s. A pioneer in this field was Dr. Murray Goodman, who applied a battery of methodologies to study the onset of structure in homooligopeptides. This article reviews some of Dr. Goodman's contributions, and reports recent studies using linear and constrained peptides corresponding to the first extracellular loop and linear peptides corresponding to the sixth transmembrane domain of a G-protein coupled receptor from the yeast Saccharomyces cerevisiae. Peptides containing 30-40 residues were synthesized using solid-phase methods and purified to near homogeneity by reversed phase high performance liquid chromatography. CD and NMR analyses indicated that the first extracellular loop peptides were mostly flexible in water, and assumed some helical structure near the N-terminus in trifluoroethanol and in the presence of micelles. Comparison of oligolysines with native loop residues revealed that three lysines at each terminus of a peptide corresponding to the sixth transmembrane domain of the alpha-factor receptor resulted in better aqueous solubility and greater helicity than the native loop residues.