Structural features and conformational equilibria of 310-helical peptides in solution by spectroscopic and molecular mechanics studies

The structural features and conformational equilibria of a series of short, linear Calpha-methylvaline [(alphaMe)Val]-based peptides in methanol were investigated by combining fluorescence resonance energy transfer measurements and molecular mechanics data. IR spectra were employed to determine their secondary structure, which exhibits an intramolecularly H-bonded, 3(10)-helix conformation that is affected by backbone distortions that are enhanced by the shortness of the main chain.     

A spectroscopic and molecular mechanics investigation on a series of AIB-based linear peptides and a peptide template, both containing tryptophan and a nitroxide derivative as probes

Linear Aib-based hexapeptides, of the general formula Ac-Toac-(Aib)(n) -Trp-(Aib)(r) -OtBu [T(Aib)(n) Trp], where n + r = 4, and Toac is a nitroxide spin-labeled C(alpha,alpha)-disubstituted glycine, were investigated by steady-state and time-resolved fluorescence measurements in different solvent media. A related peptide, i.e., cyclo-?Orn-[(Aib)(2)-Trp-(Aib)(2)-Z]-Asp-[(Aib)(2)-Toac-(Aib)(2)-+ ++OtBu ]? [T-cyclo-Trp], was also studied by the same techniques. It is a L-Orn, L-Asp diketopiperazine template, to which two Aib-based chains are covalently attached, each one containing one chromophore only, i.e., Trp or Toac. Whatever the solvent, in the former series of peptides quenching of the excited Trp exhibits three lifetime components and proceeds on a time scale from subnanoseconds to a few nanoseconds, while in the case of the template the same process occurs entirely on the nanoscale time scale, exhibiting two lifetimes only. The ir absorption spectral patterns suggest that the backbone of the peptides examined is in the 3(10)-helical conformation, as earlier determined by x-ray diffraction for T(Aib)(3)Trp in the crystal state. In all cases, the fluorescence results are satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited Trp to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process, allowing us to estimate the dynamics of the process. Molecular mechanics calculations coupled with time decay data made it possible to build up the most probable structures of these peptides in solution.     

Folding of peptides characterized by c3Val,a highly constrained analogue of valine

Using a combined chemical/chiral chromatographic approach we synthesized an N-protected derivative of (R)-c(3)Val, a severely conformationally restricted C(alpha)-tetrasubstituted alpha-amino acid characterized by a C(beta,beta)-dimethylated cyclopropane system. A set of terminally protected derivatives and model peptides (to the heptamer level), containing one or two (R)-c(3)Val residues in combination with either Aib or Gly residues, was prepared by solution methods. A detailed solution and crystal-state conformational investigation, based on Fourier transform infrared (FTIR) absorption, (1)H-NMR, and x-ray diffraction techniques, performed in comparison with a similar study on related derivatives and peptides rich in (alphaMe)Val, the prototype of C(alpha)-tetrasubstituted alpha-amino acids of this subfamily, allowed us to conclude the following: (a) c(3)Val is a good beta-bend and helix former, although less efficient than (alphaMe)Val. (b) The relationship between alpha-carbon chirality and screw sense of the folded structure formed is the same as that of (alphaMe)Val, i.e., the (R)-enantiomer has a strong left-handed bias. (c) c(3)Val seems more prone than (alphaMe)Val to fold into a gamma-bend conformation. The conformational propensities of C(beta,beta)-disubstituted Ac(3)c residues are also discussed in comparison with those of the parent cyclopropane residue.     

Distance dependency of exciton coupled circular dichroism using turn and helical peptide spacers

Porphyrins are promising chromophores for the investigation of the still unexplored area of 3-dimensional structural studies of proteins by using the exciton coupled circular dichroism (CD) method. The synthesis, conformational characterization by FTIR absorption and (1)H-NMR, and CD properties are described for a model bis-porphyrin system based on homooligo-[L-(alphaMe)Val](n) peptides as rigid spacers. In particular, the coupled CD phenomenon is experimentally detected, the intensity of which is modulated by the interchromophoric distance. These results extend and integrate those already reported with steroid, dimeric steroid, and brevetoxin bridges.     

Comparative EPR and fluorescence conformational studies of fully active spin-labeled melanotropic peptides

Similar to melanocyte stimulating hormone (alpha-MSH), its potent and long-acting analogue, [Nle(4), D-Phe(7)]alpha-MSH, when labeled with the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac), maintains its full biological potency, thus validating any comparative structural investigations between the two labeled peptides. Correlation times, calculated from the electron paramagnetic resonance signal of Toac bound to the peptides, and Toac-Trp distances, estimated from the Toac fluorescence quenching of the Trp residue present in the peptides, indicate a more rigid and folded structure for the potent analogue as compared to the hormone, in aqueous medium.     

Structural features of linear, homo-Aib-based peptides in solution: a spectroscopic and molecular mechanics investigation.

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 properties of a series of linear, homo-Aib peptides in methanol (a structure-supporting solvent) were investigated. These compounds have the general formula P(Aib)nN, where Aib is alpha-aminoisobutyric acid, N is naphthalene and P is the monomethylated protoporphyrin IX, the two latter chromophores being covalently attached to the peptide C- and N-termini, respectively, while n=3, 6 and 9. According to 1H NMR and IR spectra, the peptides investigated largely populate a 3(10)-helical structure in CDCl3, which is also a structure-supporting solvent. Both steady-state and time-resolved fluorescence measurements show a strong quenching of the N emission that parallels an increase of the P fluorescence intensity, suggesting the occurrence of long-range energy transfer from 1N* to ground-state P. Comparison of quenching efficiencies and lifetime pre-exponents with those obtained theoretically from the deepest energy minimum conformers is very satisfactory. The computed structures, built up by partially taking into account the solvent medium, exhibit a rigid, highly compact arrangement, owing to both the 3(10)-helix conformation of the backbone chain and the very few peptide-to-chromophore covalent linkages. As a result, only one or two stable conformations for each peptide were theoretically found, 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, which implies that interconversion among conformational substates of the N linkages is slower than 10 ns, corresponding to the upper limit of the energy transfer characteristic time.     

Crystal-state 3D-structural characterization of novel 3(10)-helical peptides.

The crystal-state conformations of two octapeptides, pBrBz-(D-Iva)8-OtBu (8I) and Ac-[L-(alphaMe)Val]8-OH (8II), the heptapeptide Z-[L-(alphaMe)Val]7-OH (7), the hexapeptide Z-[L-(alphaMe)Leu]6-OtBu (6) and the tetrapeptide alkylamide Z-(Aib)2-L-Glu(OMe)-L-Ala-L-Lol (5) were assessed by x-ray diffraction analyses. Two independent molecules are observed in the asymmetric unit of each L-(alphaMe)Val homo-peptide. All four homo-peptides are folded in a regular 3(10)-helical structure (only the C-terminal H-bonded conformation of the D-Iva octapeptide is distorted to a type-I beta-turn). The hydroxyl groups of the C-terminal carboxyl moieties of the two L-(alphaMe)Val homo-peptides participate in an oxy-analogue of the type-III beta-turn conformation. While the two L-(alphaMe)Val 3(10)-helices are right-handed, the D-Iva and L-(alphaMe)Leu helices are left-handed. The tetrapeptide alkylamide is 3(10)-helical at the N-terminus, but it is mixed 3(10)/alpha-helical at the C-terminus.     

(alphaMe)Nva: stereoselective syntheses and preferred conformations of selected model peptides.

Using different stereoselective chemical and chemoenzymatic approaches we synthesized the chiral, Calpha-methylated alpha-amino acid L-(alphaMe)Nva with a short, linear side-chain. A set of terminally protected model peptides to the pentamer level containing either (alphaMe)Nva or Nva in combination with Ala and/or Aib was prepared using solution methods and characterized fully. Two (alphaMe)Nva peptides were also synthesized using side-chain hydrogenation of the corresponding Calpha-methyl, Calpha-allylglycine (Mag) peptides. A detailed solution and crystal-state conformational analysis based on FT-IR absorption, 1H NMR and X-ray diffraction techniques allowed us to define that: (i) (alphaMe)Nva is an effective beta-turn and 3(10)-helix former; and (ii) the relationship between (alphaMe)Nva chirality and the screw sense of the turn/helix formed is that typical of protein amino acids, i.e. L-(alphaMe)Nva induces the preferential formation of right-handed folded structures. In more general terms, this study reinforced previous conclusions that peptides based on alpha-amino acids with a Calpha-methyl substituent and a Calpha-linear alkyl substituent are characterized by a strong tendency to fold into turn and helical structures.     

Peptide-lipid interaction monitored by spin labeled biologically active melanocortin peptides

The present work comparatively analyzes the interaction of alpha-MSH and its more potent and long-acting analog [Nle4, D-Phe7]alpha-MSH (NDP-MSH) with lipid bilayers. The peptides were spin labeled with Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at the N-terminal, as those derivatives had been previously shown to keep their full biological activity. Due to the special rigidity of the Toac covalent binding to the peptide molecule, this spin label is highly sensitive to the peptide backbone conformation and dynamics. The peptides were investigated both by the electron spin resonance (ESR) of Toac0 and the time resolved fluorescence of Trp9 present in the peptides. The Toac0 ESR of the membrane-bound peptides indicates that the two peptides are inserted into the bilayer, close to the bilayer surface, in rather similar environments. A residue titration around pKa 7.5, possibly that of His6, can be clearly monitored by peptide-lipid partition. Trp9 time resolved fluorescence indicates that the peptides, and their Toac-labeled derivatives, present rather similar conformations when membrane bound, though Trp9 in NDP-MSH, and in its Toac-labeled derivative, goes somewhat further down into the bilayer. Yet, Toac0 ESR signal shows that the Toac-labeled N-terminal of NDP-MSH is in a shallower position in the bilayer, as compared to the hormone.     

(alphaMe)Aun: a highly lipophilic, chiral, Calpha-tetrasubstituted alpha-amino acid. Incorporation into model peptides and preferred conformation.

Using a chemo-enzymatic approach we prepared the highly lipophilic, chiral, Calpha-methylated alpha-amino acid (alphaMe)Aun. Two series of terminally protected model peptides containing either D-(alphaMe)Aun in combination with Aib or L-(alphaMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT-IR absorption, 1H NMR and CD techniques, allowed us to determine the preferred conformation of this amino acid and the relationship between chirality at its alpha-carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that D-(alphaMe)Aun favors the formation of the left-handed 3(10)-helical conformation.     

Short peptide constructs mimic agonist sites of AT1R and BK receptors

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT₁R and BKRB₁ or BKRB₂ G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S–S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac¹-AngII and Toac⁰-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac³-AngII and Toac³-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT₁R and BKRB cyclic constructs based on the structure of the CXCR₄, an α-chemokine GPCR-type receptor.     

Ganglioside GM3 modulates conformation of reconstituted Ca~(2 ) -ATPase

Using steady-state fluorescence and nanosecond time-resolved fluorescence techniques, the Ca 2 -ATPase conformational changes induced by ganglioside GM3 were studied with different quenchers. The results showed that GM3 could significantly increase the lifetime of intrinsic fluorescence of Ca2 -ATPase reconstituted into proteoliposomes, and could also weaken the intrinsic fluorescence quenching by KI or hypocrellin B, HB. Further-more, by using quenching kinetic analysis of the time-resolved fluorescence, in the presence of GM3, the quenching constant (Ksv) and quenching efficiency were significantly lowered. The obtained results suggest that the oligosaccha-ride chain and the ceramide moieties of the GM3 molecule could interact with its counterparts of the Ca2 -ATPase re-spectively, thus change the conformation of the hydrophobic domain of the enzyme, making the tryptophan residues in different regions shift towards the hydrophilic-hydrophobic interface, and hence shorten the distance between the hy     

Fluorescence study of conformational properties of melanotropins labeled with aminobenzoic acid.

The native hormone alpha-melanocyte-stimulating hormone (alpha-MSH) and its more potent analog [Nle(4),D-Phe(7)]alpha-MSH (NDP-alpha MSH), labeled at the amino terminal with the fluorescent aminobenzoic acid (Abz) isomers, were examined by fluorescence methods. We observed energy transfer between the tryptophan(9) residue acting as donor and Abz as acceptor, the transfer being more pronounced to the ortho-form of the acceptor. Within the hypothesis that different peptide conformations coexist in equilibrium during the fluorescence decay, we supposed that the intensity decay was modulated by an acceptor-donor distance distribution function f(r). From the time-resolved fluorescence experimental data, we recovered the distance distribution between Abz and Trp(9), using the CONTIN program, within the framework of the F?rster resonance energy transfer model. The methodology proved to be useful to provide quantitative information about conformational dynamics of melanotropins and its dependency on the solvent. In aqueous medium, alpha-MSH has a broad Abz-Trp(9) distance distribution, reflecting the structural flexibility of the peptide. Three different distance populations could be identified in the labeled analog NDP-alpha MSH in water, indicating distinct conformational states for the synthetic peptide, compared with the native hormone. Measurements in trifluoroethanol resulted in the recovery of two Abz-Trp(9) distance populations, both for the native and the analog hormones, reflecting the decrease, induced by the solvent, of the conformational states available to the peptides.     

Slow tert-butyl ester acidolysis and peptide 3(10)-helix to alpha-helix transition in HFIP solution

We have already shown by CD and NMR techniques that the terminally protected homo-octapeptides Z (and Ac)-[L-(alphaMe)Val](8)-OtBu undergo a slow and irreversible 3(10)-helix to alpha-helix transition when dissolved in 1,1,1,3,3,3-hexafluoroisopropanol. In the present work, we find by HPLC and CD that under the aforementioned experimental conditions, a slow acidolysis of the tert-butyl ester functionality does take place affording the corresponding octapeptide free acids. The results of our combined chromatographic and spectroscopic experiments are confirmed by a comparison with the properties of independently synthesized and chemically characterized authentic compounds.     

Preferred solution conformation of peptides rich in the lipophilic, chiral, C(alpha)-methylated alpha-amino acid (alpha Me)Aoc.

The lipophilic, chiral, C(alpha)-methylated alpha-amino acid L-(alphaMe)Aoc (2-methyl-2-amino-octanoic acid) was prepared using a chemo-enzymatic approach. Two series of terminally protected model peptides, from dimer through to hexamer, containing L-(alphaMe)Aoc in combination with either Gly or Aib, were synthesized by solution methods and were fully characterized. A solution conformational analysis, based on FT-IR absorption, 1H-NMR and circular dichroism (CD) techniques, was performed with the aim at determining the preferred conformation of this novel amino acid and the relationship between chirality at its alpha-carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that L-(alphaMe)Aoc favours the formation of the right-handed 3(10)-helical conformation.     

Acid-base titration of melanocortin peptides: evidence of Trp rotational conformers interconversion

Tryptophantime-resolved fluorescence was used to monitor acid-base titration properties of alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more potent analog [Nle4, D-Phe7]alpha -MSH (NDP-MSH), labeled or not with the paramagnetic amino acid probe 2,2,6,6-tetramthylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Global analysis of fluorescence decay profiles measured in the pH range between 2.0 and 11.0 showed that, for each peptide, the data could be well fitted to three lifetimes whose values remained constant. The less populated short lifetime component changed little with pH and was ascribed to Trp g+ chi1 rotamer, in which electron transfer deactivation predominates over fluorescence. The long and intermediate lifetime preexponential factors interconverted along that pH interval and the result was interpreted as due to interconversion between Trp g- and trans chi1 rotamers, driven by conformational changes promoted by modifications in the ionization state of side-chain residues. The differences in the extent of interconversion in alpha-MSH and NDP-MSH are indicative of structural differences between the peptides, while titration curves suggest structural similarities between each peptide and its Toac-labeled species, in aqueous solution. Though less sensitive than fluorescence, the Toac electron spin resonance (ESR) isotropic hyperfine splitting parameter can also monitor the titration of side-chain residues located relatively far from the probe.     

Mixing ratio-dependent energy transfer from DNA-bound 4',6-diamidino-2-phenylindole to [Ru(1,10-phenanthroline)(2)dipyrido[3,2-a:2',3'-c]phenazine](2+)

The binding mode of Delta- and Lambda-[Ru(1,10-phenanthroline)(2)dipyrido[3,2-a:2',3'-c]phenazine](2+) ([Ru(phen)(2)DPPZ](2+)) to DNA in the presence of 4',6-diamidino-2-phenylindole (DAPI) at a low and high [DAPI]/[DNA base] ratio (0.02 and 0.20, respectively) was investigated using electric absorption and circular dichroism spectroscopy. The spectral properties of both the Delta- and Lambda-[Ru(phen)(2)DPPZ](2+) were not altered in the presence of DAPI disregarding the [DAPI]/[DNA] ratio, suggesting that the presence of DAPI in the minor groove of DNA does not affect the binding mode of the [Ru(phen)(2)DPPZ](2+) complex to DNA. The transferring excited energy of DAPI to both Delta- and Lambda-[Ru(phen)(2)DPPZ](2+) occurs through F?rster type resonance when they both spontaneously bound to DNA. At a high [DAPI]/[DNA] ratios, an upward bending curve in the Stern-Volmer plot, and a shortening the DAPI fluorescence decay time with increasing [Ru(phen)(2)DPPZ](2+) concentration were found. These results indicate that the quenching of the DAPI's fluorescence occurs through both the static and dynamic mechanisms. In contrast, the quenching mechanism at a low [DAPI]/[DNA] ratios was found to be purely static. The static quenching constant decreased linearly with respect to the [DAPI]/[DNA] ratio. Decrease in quenching efficiency can be explained by the association constant of [Ru(phen)(2)DPPZ](2+) to DNA while being within a quenchable distance from a DAPI molecule.     

Local structure in a tryptic fragment of performic acid oxidized ribonuclease A corresponding to a proposed polypeptide chain-folding initiation site detected by tyrosine fluorescence lifetime and proton magnetic resonance measurements

The effects of proline and X-Pro peptide bond conformations on the fluorescence properties of tyrosine in peptides corresponding to parts of a proposed chain-folding initiation site in bovine pancreatic ribonuclease A are examined by time-resolved and steady-state fluorescence spectroscopy. In peptides with Tyr-Pro sequences, the conformational constraints of proline on a preceding residue result in significant fluorescence quenching for both trans and cis peptide bond conformations. Small peptides containing Pro-Tyr sequences, on the other hand, do not exhibit fluorescence quenching compared to Ac-Tyr-NHMe. Studies of fluorescence decay in the tryptic fragment of performic acid oxidized ribonuclease corresponding to residues 105-124 (i.e., O-T-16) demonstrate the presence of at least two environments of the single tyrosine chromophore (in the sequence Asn113-Pro114-Tyr115). In these two (ensemble-averaged) environments, tyrosine has shorter and longer lifetimes, respectively, than in Ac-Tyr-NHMe. The fluorescence heterogeneity in O-T-16 does not correlate with X-Pro cis/trans conformational heterogeneity that can be detected by nuclear magnetic resonance (NMR) spectroscopy. Instead, the fluorescence heterogeneity in O-T-16 arises from the presence of multiple conformations with the same X-Pro peptide bond conformations which interconvert rapidly on the 1H NMR time scale (tau much less than 1 ms) but are distinguishable on the fluorescence lifetime time scale (tau greater than or equal to 1 ns). From comparisons with the tyrosine fluorescence decay of smaller synthetic peptides, it is concluded that the long-lifetime tyrosine fluorescence component of O-T-16 arises from interactions involving residues outside the Asn113-Pro114-Tyr115-Val116-Pro117 sequence, which either stabilize particular local conformations in the vicinity of Tyr115 or act directly to protect Tyr115 from efficient fluorescence quenching. The short-lifetime component of O-T-16 is also observed for the pentapeptide Ac-Asn-Pro-Tyr-Val-Pro-NHMe. The data provide evidence for a nonrandom polypeptide conformation of O-T-16 under conditions of solvent pH and temperature at which the complete disulfide-intact ribonuclease molecule is fully folded. Implications of this work for the interpretation of fluorescence-detected unfolding experiments are discussed.     

Synthesis, conformation, and bioactivity of novel analogues of the antiviral lipopeptide halovir A.

We synthesized by solution-phase methods three analogues, [L-Leu(6)-OMe], [L-(alphaMe)Leu(3), L-Leu(6)-OMe], and [L-(alphaMe)Val(4), L-Leu(6)-OMe] of halovir A. The [L-Leu(6)-OMe] analogue is known to be biologically equipotent to its naturally occurring, antiviral, lipopentapeptide amide parent compound. The preferred conformations of the L-(alphaMe)Leu- and L-(alphaMe)Val-containing analogues, with a potentially reinforced helicity, were compared with those of [L-Leu(6)-OMe] halovir A and the natural peptide itself by use of a combination of FT-IR absorption and NMR techniques. Measurements of the antiviral activities against herpes simplex virus type-1 (HSV-1) of halovir A and its three analogues were also carried out. Interestingly, the [L-(alphaMe)Val(4), L-Leu(6)-OMe] analogue exhibits the most significant activity in reducing HSV-1 infectivity, notably higher than that of halovir A itself.     

Ganglioside GM3 modulates conformation of reconstituted ca2+-ATPase

Using steady-state fluorescence and nanosecond time-resolved fluorescence techniques, the ca²⁺ ATPase conformational changes induced by ganglioside GM3 were studied with different quenchers. The results showed that GM3 could significantly increase the lifetime of intrinsic fluorescence of Ca²⁺-ATPase reconstituted into proteoliposomes, and could also weaken the intrinsic fluorescence quenching by KI or hypocrellin B, HB. Furthermore, by using quenching kinetic analysis of the time-resolved fluorescence, in the presence of GM3, the quenching constant (K_3V) and quenching efficiency were significantly lowered. The obtained results suggest that the oligosaccharide chain and the ceramide moieties of the GM3 molecule could interact with its counterparts of the ca²⁺-ATPase respectively, thus change the conformation of the hydrophobic domain of the enzyme, making the tryptophan residues in different regions shift towards the hydrophilic-hydrophobic interface, and hence shorten the distance between the hydrophilic and the hydrophobic domains, making the enzyme with a more compact form exhibit higher enzyme activity.