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The conformational analysis of [Aib 5,6 -D-Ala 8 ]cyclolinopeptide A, in the solid state and solution, has been carried out by X-ray diffraction and NMR spectroscopy. The structure of the orthorhombic form, obtained from methanol-water mixture [a=29.92 (3) A b=19.85 (3) A, c=9.90 (1) A, space group P2 1 2 1 2 1 Z=4], shows the presence of five in N-H...O=C hydrogen bonds, with formation of one C 17 ring structure, one α-turn (C 13 ), one γ-turn (C 7 ), and two β-turns (C 10 , one of type III and one of type I)

The trans-[Cl2-Pt-(H-Aib-OH)2] and trans-[Pt-(H-Aib-O−)2] complexes have been synthesized and characterized in solution and in the solid state. Their crystal and molecular structures have been determined by X-ray diffraction analyses. The complexes show a distorted planar coordination around the heavy atom. The organic moiety in the two complexes shows different conformations: in trans-[Pt-(H-Aib-O−)2] the amino acid residue, acting as a bidentate, is forced to assume an unusual conformation. On the other hand, in trans-[Cl2-Pt-(H-Aib-OH)2], in which the amino acid acts as a monodentate ligand, the α-aminoisobutyric acid assumes an extended conformation, which in general for α-monosubstituted amino acid residues coordinated to the platinum should be the preferred conformation. In both crystal structures hydrogen bonds are formed between the donor and acceptor groups with further stabilization deriving from van der Waals interactions between hydrophobic moieties.

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and solution conformational characterization of the cyclic tetrapeptide cyclo-(L-Pro-beta-Ala-L-Pro-beta-Ala). This peptide was synthesized by classical solution methods and the cyclization of the free tetrapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide (DCCI). The compound crystallizes in the orthorombic space group P2(1)2(1)2(1) from ethyl acetate. All peptide bonds are trans. The molecular conformation is stabilized by two intramolecular hydrogen bonds between the CO and NH groups of the two beta-alanine residues. These hydrogen bonds take place in a C7 structure in which both proline residues occupy the 2 position of an inverse gamma-turn. The two beta-alanine residues have a typical folded conformation (around the C alpha-C beta bond) observed in other cyclic peptides containing this residue. A detailed 1H-nmr analysis in CD3CN solution has been carried out. The molecule assumes a twofold symmetry in solution with a molecular conformation consistent with that observed in the solid state.

Cyclodextrins (CDs) are cyclic oligomers of α-1,4-linked D-glucopyranose. Due to their unique structure, marked by a chiral and hydrophobic cavity, CDs have been extensively used as chiral selectors and drug delivery systems. The functionalization both improve the CD applications and widen their use in many other fields, such as molecular recognition and enzyme mimicking. Moreover, the functionalization highly increases the metal binding properties of the CDs. This critical review is a report of recent applications concerning the CD derivatives and their metal complexes. The metal ion assists the host–guest interaction often increasing the properties of CDs to act as chiral receptors. Furthermore, it can act as a catalytic center in the mimicking of metalloenzymes based on functionalized CDs (164 references).

To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpha(v)beta(3) from alpha(v)beta(5) integrin, thus allowing the in vivo selective visualization of alpha(v)beta(3) expression by single-photon and positron emission tomography (PET) imaging.The chimeric peptide was preliminarily tested for inhibition of alpha(v)beta(3)-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpha(v)beta(3) (Kalpha(v)beta(3)) or alpha(v)beta(5) (Kalpha(v)beta(5)) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpha(v)beta(3), Kalpha(v)beta(5), U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging.Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to alpha(v)beta(3) integrin and does not cross-react with alpha(v)beta(5). In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing alphavbeta3 and fails to accumulate in those expressing alpha(v)beta(5) integrin. When 18F-labeled truncated derivative was used for PET imaging, alphavbeta3- and alpha(v)beta(5)-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins.Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alphavbeta5 integrin, allows highly selective alphavbeta3 expression imaging and monitoring.

The conjugates of beta-cyclodextrins with R- or with S-Etodolac were characterized by NMR spectroscopy, and S-Etodolac alone was characterized by X-ray diffraction analysis. In solution, the R-Etodolac conjugate is soluble in water; the other epimer shows a very low solubility. The NMR characterization of the R-Etodolac conjugate in D(2)O shows that, in aqueous solution, the Edotolac moiety is self-included in the cavity, while the NMR characterization in MeOH of both conjugates underlines that, in this solvent, the Etodolac moiety is not included in the CD cavity. The X-ray structure determination of the S-Etodolac conjugate reveals a "sleeping swan"-like shape, with the covalently bonded Etodolac moiety being folded with the 8-ethyl group inserted inside the hydrophobic cavity of the beta-CD ring. The terminal methyl group of the 8-ethyl group enters the centre of cavity from the side of the primary hydroxyl groups and is buried inside the beta-CD macrocycle. The terminal methyl group is positioned at a distance of 1.06 A from the O(4) plane in the side of the primary hydroxyl groups. In addition to van der Waals interactions between the hydrophobic ethyl group and the beta-CD cavity, the folded conformation is further stabilized by one intramolecular H-bond involving the indole N-H group and the primary hydroxyl group of the glucose unit 7. Along the b axis, the beta-CD molecules are arranged in columns; the macrocycles form a herring bone pattern, so that the cavity of each beta-CD molecule is closed at each end by neighboring molecules. Within the layers, the beta-CD macrocycles are held together by a complicated intermolecular hydrogen bond network, in which numerous water molecules and hydroxyl groups are involved.

The use of peptides as targeting tools has been validated in different applications. In particular radiolabelled peptides with adequate stability, receptor-binding properties and biokinetic behaviour have been investigated as an important class of radiopharmaceuticals for cancer pathology imaging and therapy. This review focuses on recent progress in design and synthetic modifications of small biologically active peptides used in diagnosis and therapy. In particular, we report the current development and optimization of suitable peptides for targeting three relevant biological receptors (CCK, somatostatin, and integrin receptors) involved in specific tumour diseases.

Purpose: To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate αvβ3 from αvβ5 integrin, thus allowing the in vivo selective visualization of αvβ3 expression by single-photon and positron emission tomography (PET) imaging. Experimental Design: The chimeric peptide was preliminarily tested for inhibition of αvβ3-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing αvβ3 (Kαvβ3) or αvβ5 (Kαvβ5) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kαvβ3, Kαvβ5, U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Results: Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to αvβ3 integrin and does not cross-react with αvβ5. In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing αvβ3 and fails to accumulate in those expressing αvβ5 integrin. When 18F-labeled truncated derivative was used for PET imaging, αvβ3- and αvβ5-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Conclusion: Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with αvβ5 integrin, allows highly selective αvβ3 expression imaging and monitoring. (Clin Cancer Res 2009;15(16):5224–33)

Introduction Angiogenesis, the process whereby new capillaries are formed by outgrowth from existing microvessels, is required for tumor growth and metastasis [1,2]. The transmembrane cell-surface receptor αvβ3 has recently received increasingly attention, because of the critical role in tumor associated angiogenesis and metastasis formation. The restricted expression of integrin αVβ3 during tumor growth, invasion, and metastasis presents an interesting target for both detection and treatment of solid tumors [3]. Targeting αVβ3 with radiolabelled ligands may provide information about the receptor status and enable the planning and the monitoring of therapeutic approaches. Recently we developed a novel αVβ3 antagonist that showed a high selectivity for the receptor [4]. Adhesion assays, competitive binding assays and cross-linking experiments performed in human erythroleukemia K562 cells, stably cotransfected with cDNA of αv or αIIb and β3, demonstrated the high selectivity for αvβ3 integrin [4]. Starting from these evidences RGDechi was covalently bound to the chelating agent DTPA (Diethylene Triamine Pentaacetic Acid ) able to give stable complexes of radionuclides such as In and Y. The final goal has been to obtain a radiolabelled compound to be used in nuclear medicine as diagnostic and therapeutic agent. In particular DTPA-RGDechi has been labelled with In and used in SPECT (Single Photon Emission Computed Tomography) for diagnostic purpose. Moreover, the ligand RGDechi has been labelled with F for micro-PET in order to evaluate the use of this peptide for in vivo imaging.

Cyclodextrins (CDs) are cyclic oligomers of alpha-1,4-linked D-glucopyranose. Due to their unique structure, marked by a chiral and hydrophobic cavity, CDs have been extensively used as chiral selectors and drug delivery systems. The functionalization both improve the CD applications and widen their use in many other fields, such as molecular recognition and enzyme mimicking. Moreover, the functionalization highly increases the metal binding properties of the CDs. This critical review is a report of recent applications concerning the CD derivatives and their metal complexes. The metal ion assists the host-guest interaction often increasing the properties of CDs to act as chiral receptors. Furthermore, it can act as a catalytic center in the mimicking of metalloenzymes based on functionalized CDs (164 references).

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO) contains three disulphide bridges linking residues C(41)XXC(44), C(155)XXC(158), C(173)XXXXC(178). To get information on the role played by these cross-links in determining the structural and functional properties of the protein, we performed site-directed mutagenesis on Cys residues and investigated the changes in folding, stability and functional features of the mutants and analysed the results with computational analysis. The reductase activity of SsPDO and its mutants was evaluated by insulin and thioredoxin reductase assays also coupled with peroxiredoxin Bcp1 of S. solfataricus. The three-dimensional model of SsPDO was constructed and correlated with circular dichroism data and functional results. Biochemical analysis indicated a key function for the redox site constituted by Cys155 and Cys158. To discriminate between the role of the two cysteine residues, each cysteine was mutagenized and the behaviour of the single mutants was investigated elucidating the basis of the electron-shuffling mechanism for SsPDO. Finally, cysteine pK values were calculated and the accessible surface for the cysteine side chains in the reduced form was measured, showing higher reactivity and solvent exposure for Cys155.

The specific inhibition of serine proteinases, which are crucial switches in many important physiological processes, is of great value both for basic research and for therapeutic applications. In this study, we report the molecular cloning of the sso0767 gene from Sulfolobus solfataricus, and the functional characterization of its product, SsCEI, which represents the first archaeal phosphatidylethanolamine-binding protein (PEBP)-serine proteinase inhibitor, reported to date. SsCEI is a monomer protein with a molecular mass of 19.0 kDa and a pI of 6.7, which is able to inhibit the serine proteases alpha-chymotrypsin and elastase with K(i) values of 0.08 and 0.1 microM, respectively. Moreover SsCEI is extremely resistant to both thermal inactivation and proteolytic attack suggesting compact folding of the protein. Within the I51 family, the archaeal inhibitor shows strong similarity to the human and murine members. The three-dimensional model of SsCEI revealed a general beta-fold and the presence of an anion-binding pocket, the hallmark of the PEBP family. Moreover SsCEI binds the cognate proteases according to a common, substrate-like standard mechanism. Point mutation experiments supported the prediction of the protease-binding site located on the surface at the C- terminal region of the protein. Interestingly, searches based on preidentified structural reactive loop motifs revealed the occurrence of a sequence (T123-N130) that is not represented in all serine-protease inhibitor families. This unique motif may provide new insights into both the inhibitor/protease binding mode and the specific biological functions of SsCEI within the PEBP family.

In the present work, we report the synthesis and the characterization of dab PNA hexamers with diaminobutyric acid backbone of D- or/and L-configuration. In particular, the four nucleo-amino acids we synthesized, D- and L-diaminobutyryl adenines and D- and L-diaminobutyryl thymines, were used in various combinations to assemble the following oligomers: H-G-(t( L-dab))(6)-K-NH(2), H-G-(t( D-dab))(6)-K-NH(2), H-G-(a( L-dab))(6)-K-NH(2), H-G-(t( L-dab)-t( D-dab))(3)-K-NH(2), H-G-(a( L-dab)-a( D-dab))(3)-K-NH(2), H-G-(a( L-dab)-t( D-dab))(3)-K-NH(2). By using CD and UV spectroscopies, we investigated the ability of complementary dab PNA strands to bind to each other. We found that binding occurs only between oligomers with backbone of alternate configuration [(t( L-dab)-t( D-dab))(3)/(a( L-dab)-a( D-dab))(3) and (a( L-dab)-t( D-dab))(3)/(a( L-dab)-t( D-dab))(3)] and implies cooperative hydrogen bonds and base stacking. Furthermore, interesting properties relative to the self-complementary oligomer (a( L-dab)-t( D-dab))(3) forming palindromic complexes emerged from preliminary dynamic light-scattering experiments that suggested the formation of multimeric aggregates. These results, together with the high serum stability of the DABA-based oligomers, as shown by HPLC analysis, encourage us to further study dab PNAs as new self-recognizing bio-inspired polymers, to develop new nanomaterials in biotechnological and biomedical applications.

The conformation of cyclolinopeptide A [c-(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val)], a naturally occurring peptide with remarkable cytoprotective activity, has been investigated by means of molecular dynamics simulations in various molecular environments. Structural and dynamical properties have been analyzed and compared with those experimentally determined. A detailed analysis of hydrogen bonds is reported.

Blends of poly(ethylene oxide)/poly(ethylene-co-vinyl acetate) (PEO/EVAc-1) have been prepared by casting from a common solvent. The miscibility of the mixture is studied by using differential scanning calorimetry (d.s.c.) and dilatometry. The blends, as obtained, show a single, composition-dependent, glass transition that fits the Fox equation well, indicating the presence of a homogeneous amorphous phase. Some blends phase-separate on heating. The cloud-point curve of the system is drawn by detecting on the d.s.c. thermograms one or two glass transition temperatures depending on whether or not phase separation takes place at a fixed annealing temperature. The cloud-point curve is very skew, with a maximum at around 80 wt% PEO content and t = 210 ± 10°C. The theoretical approach of group contributions and solubility parameters predicts no miscibility for the system under investigation, whereas a lower critical solution temperature is predicted by Flory's equation-of-state theory. Using values of X12 interactional parameter, derived from the experimental thermal expansion coefficients of the blends, a simulated spinodal phase boundary is obtained.

The molecular basis of sweet taste was investigated by carrying out the crystal state conformational analysis by X-ray diffraction of the following dipeptide taste ligands: N-3,3-dimethylbutyl-aspartyl-phenylalanine methyl ester, I (N-DMB-Asp-Phe-OMe), its sodium salt (N-DMB-Asp-Phe-ONa). II. aspartyl-D-2-aminobutyric acid-(S)-α-ethylbenzylamide, III (Asp-D-Abu-(S)-α-ethylbenzylamide), aspartyl-N-((2,2,5,5-tetramethylcyclopentanyl)-carbonyl)-(R)-1, 1-diamino-ethane. IV (Asp-(R)-gAla-TMCP), and aspartyl-D-valine-(R)-α-methoxymethylbenzyl amide, V (Asp-D-Val-(R)-α-methoxymethylbenzylamide). With the exception of the sodium salt II, all compounds are sweet-tasting, showing in some cases considerable potency enhancement with respect to sucrose. The results of this study confirm the earlier model that an 'L-shape' molecular array is essential for eliciting sweet taste for dipeptide-like ligands. In addition, it was established that (i) substitution of the N-terminal group does not inhibit sweet taste, if its zwitterionic character is maintained; (ii) a hydrophobic group located between the stem and the base of the L-shape could be responsible for sweetness potency enhancement, as found in I, III and IV; in fact, the extraordinary potency of the N-alkylated analogue I would support a model with an additional hydrophobic binding domain above the base of the 'L'; (iii) removal of the methyl ester at the C-terminus of compound I with the salt formation gives rise to the tasteless compound II; (iv) for the first time all possible side-chain conformers (g-, g+ and t) for the N-substituted aspartyl residue were observed; and (v) a retro-inverso modification, incorporated at position 2 of the dipeptide chain, confers greater flexibility to the molecule, as demonstrated by the contemporary presence of six conformationally distinct independent molecules in the unit cell and yet sweet taste properties are maintained, as found in IV. © 1998 European Peptide Society and John Wiley & Sons, Ltd.

CLA, a natural, highly hydrophobic cyclic nonapeptide with sequence c(Pro(1)-Pro(2)-Phe(3)-Phe(4)-Leu(5)-Ile(6)-Ile(7)-Leu(8)-Val(9)-), isolated from linseed oil, was found to possess a strong immunosuppressive activity comparable, in low doses, with that of CsA, with a mechanism that depends on the inhibition of the interleukin-1 and interleukin-2 action. Structural analysis of CLA and its related compounds has underlined that the presence of the tetrapeptide Pro-Pro-Phe-Phe sequence, the Pro-Pro cis amide bond, and the 'edge-to-face' interaction are possible important features for the immunosuppressive activity of CLA. To evaluate the role and significance of 'edge-to-face' interaction in the process of molecular recognition by receptors, we have synthesised three linear precursors and three cyclic analogues of CLA, in which one or both Phe residues have been replaced by beta(3)Phe residues. A conformational analysis by NMR in CD(3)CN/H(2)O mixture has been carried out on the CLA analogue, in which Phe(3) has been replaced by a betaPhe, to study the influence of the mutation on the three-dimensional structure. All linear and cyclic CLA analogues containing betaPhe have been tested in the humoral and cellular immune response in vivo assays in mice. The peptide activities have been compared with CsA, as a reference drug.

The effects of hydrostatic pressure on the structure and stability of porcine odorant-binding protein (pOBP) in the presence and absence of the odorant molecule 2-isobutyl-3-methoxypyrazine (IBMP) were studied by steady-state and time-resolved fluorescence spectroscopy as well as by molecular dynamics simulation. The authors found that the application of moderate values of hydrostatic pressure to pOBP solutions perturbed the microenvironment of Trp(16) and disrupted its highly quenched complex with Met(39). In addition, compared with the protein in the absence of IBMP, the MD simulations experiments carried out at different pressures highlighted the role of this ligand in stabilizing the Trp(16)/Met(39) interaction even at 2000 bar. The obtained results will assist for the tailoring of this protein as specific sensing element in a new class of fluorescence-based biosensors for the detection of explosives.

Acid-catalysed condensation of tryptophan with different α-amino aldehyde derivatives has been explored as a useful route to the synthesis of novel amino acid derived heterocycles and peptidomimetic scaffolds. By this approach, compounds containing a tetrahydro-β-carboline and a novel octahydropyrrolo[3′,2′:3,4]pyrrolo[2,3-b]indole system have been efficiently synthesized. Here we report the characterization of these new compounds and preliminary studies of the reactivity of the tetrahydro-β-carboline system.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Correction for ‘Solid phase synthesis of TyrT, a thymine–tyrosine conjugate with poly(A) RNA-binding ability’ by Giovanni N. Roviello et al., RSC Adv., 2016, 6, 27607–27613.

Mixtures of poly(ethylene oxide)/atactic poly(methyl methacrylate) (PEO/At-PMMA) blends and poly(ethylene oxide)/poly(ethylene-co-vinylacetate) (PEO/EVAc-1) blends were prepared by casting from a common solvent and were found miscible. The applications of the Solubility Parameter Theory, Patterson Theory and the Equation of State Theory of Flory are reported. The miscibility predictions obtained are discussed and compared to experimental miscibility evidence.

The solid state conformational analysis of the ionophoric homodetic bicyclic cyclo(Glu-Leu-Pro-Gly-Lys-Leu-Pro-Gly)cyclo(1γ-5ϵ)Gly (BCP3) has been carried out by x-ray diffraction. It crystallizes with 4.5 molecules of water per peptide molecule in the monoclinic system, space group P21, with a = 11.425 A, b = 16.616 A, c = 13.931 A, β = 109.24°, and Z = 2. The structure has been determined by direct methods and refined to an R factor of 0.061 for 2448 observed reflections. The structure characterized by all trans peptide bonds is stabilized by three intramolecular hydrogen bonds: a type II β-turn, a mixed type I-type III β-turn, and a pseudo γ-turn, which involves the side chain CO and the main-chain NH groups of the Glu1 residue. The resulting globular molecule presents a rather hydrophilic surface with most of the CO groups available to hydration of the solvent molecules, which are linked through hydrogen bonds of the NH … O or OH … O types in a complicated H-bonding scheme. The conformation observed in the solid state is rather different from the conformation proposed in solution for both the free and the Ca2+-complexed BCP3 molecule.

Peptide nucleic acids are oligonucleotide mimics characterised by high chemical and enzymatic stability, high specificity and affinity toward complementary DNA/RNA. The lack of charge and polar groups in the backbone decrease their solubility in aqueous environment and their ability to cross cell membranes, reducing their performance in in vivo applications. To improve solubility, increase affinity and specificity of binding and to control recognition between nucleic acids, several analogues bearing modifications on the nucleobase, nucleobase-backbone linker and on the backbone were synthesised. This paper describes the synthesis and applications of Peptide nucleic acid analogues and discusses the potential of analogues for which no application is reported.

A library of cyclic CCK8 analogues, containing unnatural amino acids in the peptide sequence, is prepared using solid-phase synthesis. The structure of these cyclic peptides is based on a previously synthesised compound, cyclo -CCK8, selective for CCK 1 receptor. Structure–activity investigations are performed by evaluating the binding properties of the new analogues. In particular, the binding ability of the cyclic CCK8 analogues is tested by nuclear medicine studies on cell line transfected with CCK 1 receptor. Compounds named cyclo -A4– cyclo -A7 show binding constant in the range 6.0–8.0 μM, with an improved affinity over the previous described cyclo -CCK8, but almost comparable IC 50 values among new analogues towards CCK 1 were obtained.

The complete genome of the psychrophilic bacteria Pseudoalteromonas haloplanktis TAC 125, recently published, owns a gene coding for a putative esterase activity corresponding to the ORF PSHAa1385, also classified in the Carbohydrate Active Enzymes database (CAZY) belonging to family 1 of carbohydrate esterase proteins. This ORF is 843 bp in length and codes for a protein of 280 amino acid residues. In this study we characterized and cloned the PSHAa1385 gene in Escherichia coli. We also characterized the recombinant protein by biochemical and biophysical methodologies. The PSHAa1385 gene sequence showed a significant homology with several carboxyl-esterase and acetyl-esterase genes from gamma-proteobacteria genera and yeast. The recombinant protein exhibited a significant activity towards pNP-acetate, alpha-and beta-naphthyl acetate as generic substrates, and 4-methylumbelliferyl p-trimethylammonio cinnamate chloride (MUTMAC) as a specific substrate, indicating that the protein exhibits a feruloyl esterase activity that it is displayed by similar enzymes present in other organisms. Finally, a three-dimensional model of the protein was built and the amino acid residues involved in the catalytic function of the protein were identified.

The D-galactose/D-glucose-binding protein (GGBP) from E. coli serves as an initial component for both chemotaxis toward glucose and high-affinity active transport of the sugar. In this work, we have used phosphorescence spectroscopy to investigate the effects of glucose and calcium on the dynamics and stability of GGBP. We found that GGBP exhibits a phosphorescence spectrum composed of two energetically distinct 0,0-vibrational bands centered at 404.43 and 409.61 nm; the large energy separation between them indicates two classes of chromophores making distinct dipolar interactions with their surrounding. Interestingly, the high-energy spectral component (404.43 nm) is one of the bluest spectra reported to date in proteins. Considering the ground state dipole direction, low-energy configurations for the indole side chain in proteins leading to blue-shifted spectra can arise from negative charges in proximity to the imidazole-ring nitrogen and/or positive charges near C4-C5 of the benzene ring. Among the five tryptophan residues of GGBP, Trp-284, located at the N-terminal domain of the protein, and Trp-183, located in the protein hinge region, make strong attractive charge interactions with surrounding side chains. Regarding Trp-284, the indole ring nitrogen is in contact with the negative charge of the Asp-267, whereas Trp-183 is next to the Glu-149 residue. In the latter, the ground state energy is further lowered by the proximity of the Arg-158 to the negative end (near C6) of the indole dipole. Regarding the red spectral component (409.61 nm), it is more intense than the blue component, presumably because more residues contribute to it. lambda 0,0 is typical of environments that are weakly polar or characterized by charges positioned near 90 degrees from the ground state dipole direction (the case of W195 and W127). The binding of glucose modifies the phosphorescence lifetime values as well as the spectrum of GGBP, shifting the blue band 0.54 nm to the blue and the red band 1 nm to the red. Finally, the removal of the calcium from GGBP structure causes variations in lifetime values and spectral shifts similar to those induced by glucose binding to the native protein. Aided by a detailed inspection of the three-dimensional structure of GGBP, these results contribute to a better understanding of the structure/function relationship of this protein.

We demonstrate here that tetracycline (TC) can strongly interact (KD′ = 189 ± 7 nM) with model peptides derived from the C-terminal globular domain of the prion protein, hPrP [173-195], and that interaction concerns residues within the C-terminal half of the helix 2, a short region previously indicated as endowed with ambivalent conformational behavior and implicated in PrP conversion to the β-sheet-rich, infective scrapie variant. Data have been confirmed by binding studies with the N-terminal truncated 180-195 variant that displays a dissociation constant of 483 ± 30 nM. Remarkably, TC does not influence the structure of the N-terminally fluoresceinated peptides that both show α-helical conformations. Docking calculations and molecular dynamics simulations suggest a direct, strong interaction of the antibiotic with exposed side chain functional groups of threonines 190-193 on the solvent-exposed surface of helix 2. Proteins 2007 © 2006 Wiley-Liss, Inc.

One of the most exciting area of research in drug design concerns with the synthesis and 3D-structural characterisation of molecules containing peptidomimetics, since they can be expected to possess similar biological effects as their natural peptide counterparts, so that they could be used as therapeutics, with the potential, added advantages of higher metabolic stability, enhanced interactions with the receptor, and improved pharmacokinetic properties. In this review, we report the structural properties of the main constrained non coded aminoacids as examples of peptidomimetics or molecular tools able to induce specific conformations in bioactive peptide analogues.

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.

The interaction between the 1-47 N-terminus of the CCK(1)-R and the anthranilic acid based antagonists has been investigated by fluorescence spectroscopy. These antagonists interact with W39 of the N-terminal domain of the CCK(1)-R like that of the endogenous ligand CCK-8. This specific interaction was not found in other nonpeptide ligands of the CCK(1)-R. Conformational studies, using NMR and energy minimization procedures, have allowed formulation of a new hypothesis on the CCK(1)-R binding mode of the anthranilic antagonists.

Linear and cyclic cyclolinopeptide A (CLA) analogues containing α-hydroxymethyl-leucine (HmL) in positions 1, 4, and 1&4, and α-hydroxymethylvaline (HmV) in position 5, were synthesized by the solid-phase peptide strategy and cyclized with the 1-Ethyl-3-(3- dimethylaminopropyl)-carbodiimide/1-hydroxy-7-azabenzotriazole (EDC/HOAt) reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation assays (LPA). Only HmL-containing peptides demonstrated at about 25% lower immunosuppressive activity, but they are four times more soluble in water solutions than the native CLA. It seems from the LPA results that peptide [(HmL4)CLA] is the most promising for further studies. This peptide was characterized in solution, at room temperature in CDCl 3, and the conformation compared with that observed for CLA in the solid state. © 2004 Wiley Periodicals, Inc.

Sp1 regulates the activation of many genes involved in tumor growth, apoptosis, and angiogenesis. We have previously shown the involvement of Sp1 in the up-regulation of urokinase receptor (uPAR) expression, a key molecule in tumor invasion and metastasis. Here, we investigated whether a marked down-regulation of Sp1 activity may inhibit uPAR expression and migration ability of MDA-MB-231 breast cancer cells. To this end, we tested the decoy ability of a novel peptide nucleic acid (PNA)–DNA chimera which carries a central DNA strand, containing Sp1-binding sequence, covalently linked to two PNA fragments at both ends (PNA–DNA–PNA, PDP). The chimera was synthesized, annealed with complementary DNA (PDP–DNA), and then tested for its ability to bind Sp1 both in vitro and in living MDA-MB-231 breast cancer cells in the presence of urokinase (uPA). This PDP–DNA decoy molecule efficiently competes for the binding to endogenous Sp1 in nuclear extracts, and upon transfection with liposomal vectors, causes a marked decrease of available Sp1 in both untreated and uPA-treated MDA-MB-231 cells. Accordingly, both uPA-dependent enhancement of uPAR expression and cell migration were strongly reduced in transfected cells. Interestingly, a detectable inhibitory effect is also observed in breast cancer cells exposed to PDP–DNA in the absence of transfection reagents. Finally, the inhibitory effect of PDP–DNA appeared to be stronger than that observed with oligonucleotides carrying Sp1 consensus sequence. Our findings show that this novel PNA–DNA chimera, containing Sp1 consensus sequence, effectively inhibits Sp1 activity, uPAR expression, and motility of breast cancer cells indicating its potential therapeutic use to prevent tumor dissemination.

Peptide nucleic acids (PNAs)-DNA chimeras have been recently described as DNA mimics constituted of a part of PNA and of a part of DNA. We have demonstrated that double stranded molecules based on PNA-DNA chimeras bind to transcription factors in a sequence-dependent manner. Accordingly, these molecules can be used for transcription factor decoy (TFD) pharmacotherapy. Effects of double stranded PNA-DNA chimeras targeting NF-kappaB and Sp1 were determined on in vitro cultured human cells and were found to be comparable to those observed using double-stranded DNA decoys. The TFD molecules based on PNA-DNA chimeras can be further engineered by addition of short peptides facilitating cell penetration and nuclear localization. Therefore, these engineered molecules could be of great interest for in vivo experiments for non-viral gene therapy of a variety of diseases, including neoplastic and viral diseases, for which the TFD approach has been already demonstrated as a very useful strategy.

The conformational features of a peptide derived by the 10-30 sequence of the mitochondrial domain of AKAP121 [Ac-IXKKPLALPGMLALLGWWWFFSRKKX 2 5 -NH 2 (X = β-Ala)] in water and in a water/triflouroethanol (TFE) mixture at 298 K have been determined by NMR and CD spectroscopy. Backbone clustering analysis of NMR-derived structures led to the identification of a single representative structure in water/TFE. The structure of the peptide consists mainly of an α-helix, whose core is the region 7-23, with a less ordered N-terminal part. These data are confirmed by CD analysis. It is noteworthy that the high hydrophobic Trp 1 6 -Phe 2 0 segment, that might also mediate interaction with tubulin, is organized in an α-helical wheel. Our conformational data can be the starting point for the development of highly selective peptides that interfere with the biological function of the Protein Kinase A scaffold protein AKAP121.

Peptide nucleic acids (PNAs) are DNA mimics constituted by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and are resistant to both nucleases and proteases. While applications of PNAs as antisense and antigene molecules have been described, PNA/DNA and PNA/PNA hybrids are not useful for transcription factor decoy (TFD) pharmacotherapy. By contrast, PNA-DNA-PNA (PDP) chimeras, constituted of sequential PNA, DNA and PNA stretches, are potent decoy molecules in vitro. Interestingly, PDP-based decoys a) are more soluble than PNAs, b) are more resistant than synthetic oligonucleotides to enzymatic activity present in cellular extracts and serum and c) can be delivered with liposomes. In the present study we demonstrated that double-stranded PNA-DNA-PNA chimeras targeting NF-kappaB transcription factors induce apoptosis of human primary osteoclasts. Our data suggest that PDP-based induction of osteoclast apoptosis could be a therapeutic approach for disorders in which bone resorption is inappropriately excessive.

Peptide nucleic acids (PNAs) are DNA mimics constituted by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and are resistant to both nucleases and proteases. While applications of PNAs as antisense and antigene molecules have been described, PNA/DNA and PNA/PNA hybrids are not useful for transcription factor decoy (TFD) pharmacotherapy. By contrast, PNA-DNA-PNA (PDP) chimeras, constituted of sequential PNA, DNA and PNA stretches, are potent decoy molecules in vitro. Interestingly, PDP-based decoys a) are more soluble than PNAs, b) are more resistant than synthetic oligonucleotides to enzymatic activity present in cellular extracts and serum and c) can be delivered with liposomes. In the present study we demonstrated that double-stranded PNA-DNA-PNA chimeras targeting NF-kappaB transcription factors induce apoptosis of human primary osteoclasts. Our data suggest that PDP-based induction of osteoclast apoptosis could be a therapeutic approach for disorders in which bone resorption is inappropriately excessive.

No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature.

The three-dimensional structure and the conformational behavior of a para-benzoylphenylalanine (Bpa) contg. trichogin dodecapeptide analog were evaluated using an X-ray diffraction anal. Results strongly support the view that, despite the unquestionable usefulness of the Bpa residue as a photoprobe, caution is needed in extrapolating the anal. results of photo crosslinking expts. and photophys. data to Bpa proximity, i.e., to protein mapping and intramol. distances, resp. It is also important to remember that peptide ligands quite often show a much reduced conformational flexibility when bound to a protein receptor site. The Bpa residue adopts a backbone conformation close to the b-sheet structure, and more importantly, its side chain is rigidified to the g- conformation because the bulky arom. residue occupies a largely hydrophobic patch in the interior of the enzyme.

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton. They bind DNA and RNA with high specificity and selectivity, leading to PNA–RNA and PNA–DNA hybrids more stable than the corresponding nucleic acid complexes. The binding affinity and selectivity of PNAs for nucleic acids can be modified by the introduction of stereogenic centers (such as d -Lys-based units) into the PNA backbone. To investigate the structural features of chiral PNAs, the structure of a PNA decamer containing three d -Lys-based monomers (namely H-GpnTpnApnGpnAdlTdlCdlApnCpnTpn-NH 2 , in which pn represents a pseudopeptide link and dl represents a d -Lys analogue) hybridized with its complementary antiparallel DNA has been solved at a 1.66-Å resolution by means of a single-wavelength anomalous diffraction experiment on a brominated derivative. The d -Lys-based chiral PNA–DNA (LPD) heteroduplex adopts the so-called P-helix conformation. From the substantial similarity between the PNA conformation in LPD and the conformations observed in other PNA structures, it can be concluded that PNAs possess intrinsic conformational preferences for the P-helix, and that their flexibility is rather restricted. The conformational rigidity of PNAs is enhanced by the presence of the chiral centers, limiting the ability of PNA strands to adopt other conformations and, ultimately, increasing the selectivity in molecular recognition.