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S Mammi

Publications and source records attributed to S Mammi.

At least 19 recordsLinked to original sources
PubMed2005

Conformational studies of Aib-rich peptides containing lactam-bridged side chains: evidence of 3(10)-helix formation.

Aib-rich side-chain lactam-bridged oligomers Ac-(Glu-Aib-Aib-Lys)n-Ala-OH with n = 1,2,3 were designed and synthesized as putative models of the 3(10)-helix. The lactam bridge between the side chains of L-Glu and L-Lys in (i)--(i + 3) positions was introduced in order to enhance the structural preference toward the right-handed 3(10)-helix. The conformational properties of the three peptides were studied in trifluoroethanol (TFE) solution by CD, NMR, and computer simulations. The structural information was derived mainly from the analysis of nuclear Overhauser effect spectroscopy spectra. The presence of alpha H(i)-HN(i + 2) and of alpha H(i)-HN(i + 3) connectivities and the absence of alpha H(i)-HN(i + 4) connectivities indicate that these peptides fold into a 3(10)-helix rather than into an alpha-helix. Based on these conformational features, stereospecific assignment of the Aib methyl groups was possible. The results of such experiments and of the subsequent distance geometry and restrained molecular dynamics simulations reveal a marked preference of these peptides for 3(10)-helix. The CD spectra of these peptides indicate that the helix content increases upon chain elongation. The CD spectrum of the trimer is characterized by a negative band at 200 nm and by a weak positive band around 220 nm. The CD spectrum in TFE is different from that observed in aqueous solution in the presence of SDS micelles, reported in our previous work, and from those reported by a different research group for 3(10)-helical peptides. A possible reason for these differences could rest in the presence of different equilibria of the conformer populations of the various peptides in different solvent systems.

Amino Acid Sequence↗
PubMed2005

Bioactive N-terminal undecapeptides derived from parathyroid hormone: the role of alpha-helicity.

The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N-terminal fragments presenting the principal activating domain such as PTH(1-11) and PTH(1-14) with helicity-enhancing substitutions yield potent analogues with PTH(1-34)-like activity. To further investigate the role of alpha-helicity on biological potency, we designed and synthesized by solid-phase methodology the following hPTH(1-11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix-promoting C(alpha)-tetrasubstituted alpha-amino acids alpha-amino isobutyric acid (Aib), 1-aminocyclopentane-1-carboxylic acid (Ac(5)c) and 1-aminocyclohexane-1-carboxylic acid (Ac(6)c): Ac(5)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (I); Aib-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (II); Ac(6)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (III); Aib-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (IV); Aib-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (V); S-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (VI), S-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (VII); Ac(5)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (VIII); Ac(6)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (IX); Ac(5)c-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (X); Ac(6)c-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (XI). All analogues were biologically evaluated and conformationally characterized in 2,2,2-trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I-V, which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand-stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III, VI and VII are very weakly active and analogues IV, V, VIII-XI are inactive. The most potent analogue, I exhibits biological activity 3500-fold higher than that of the native PTH(1-11) and only 15-fold weaker than that of the native sequence hPTH(1-34). Remarkably, the two most potent analogues, I and II, and the very weakly active analogues, VI and VII, exhibit similar helix contents. These results indicate that the presence of a stable N-terminal helical sequence is an important but not sufficient condition for biological activity.

Amino Acid Sequence↗
PubMed2003

Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing alpha-amino-iso-butyric acid (Aib) residues.

The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.

Amino Acid Sequence↗
PubMed2003

Conformational and biological characterization of human parathyroid hormone hPTH(1-34) analogues containing beta-amino acid residues in positions 17-19.

The N-terminal 1-34 fragment of parathyroid hormone (PTH) elicits the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(18) upon biological function, we synthesized and characterized the following human (h) PTH(1-34) analogues containing beta-amino acid residues: [analogues: see text]. Biological activity and binding affinity of analogue I are one order of magnitude lower than those of the parent compound. In analogue II, both binding affinity and biological activity are partially recovered. Analogues III and V have no binding affinity and very low biological activity. Both bioactivity and binding affinity are partially recovered in analogue IV. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, 2D-nuclear magnetic resonance and molecular dynamics calculations. The results confirmed the presence in all analogues of two helical segments located at the N-terminal and C-terminal sequences. The insertion of beta-amino acid residues around position 18 does not cause appreciable conformational differences in the five analogues. The differences in biological activity and binding affinity among the five analogues cannot be related to structural differences in the membrane mimetic environment reported in this study. Our results stress the importance of the side-chain functionalities in the sequence 17-19 for biological function.

Amino Acid Substitution↗
PubMed2002

Structure-function studies of analogues of parathyroid hormone (PTH)-1-34 containing beta-amino acid residues in positions 11-13.

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with beta-amino acid residues around the putative hinge located at position 12: I. [Nle(8,18),beta-Ala(11,12),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); II. [Nle(8,18),beta-Ala(12,13),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); III. [Nle(8,18),beta-Ala(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); IV. [Nle(8,18),beta-hLeu(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); V. [Nle(8,18),beta-Ala(12), Nal(23),Tyr(34)]bPTH-(1-34)NH(2); VI. [Nle(8,18),beta-Ala(13), Nal(23),Tyr(34)]bPTH-(1-34)NH(2) (beta-hLeu = beta-homo-leucine; beta-Ala = beta-alanine; Nal = L-2-naphthyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12, or 13 in analogues III-VI does not favor a disordered structure in this portion of the sequence.

Amino Acid Sequence↗
PubMed2001

Aib-rich peptides containing lactam-bridged side chains as models of the 3(10)-helix.

Aib-rich side chain lactam-bridged oligomers with n =1, 2, 3, were designed and synthesized as putative models of the 3(10)-helix. These peptides were conformationally characterized in aqueous solution containing SDS micelles by CD, NMR, and computer simulations. The lactam bridge between the side chains of L-Glu and L-Lys in (i) and (i+3) positions was introduced in order to enhance the conformational preference toward the right-handed 3(10)-helix. The NMR results clearly indicate that there is an increase of 3(10)-helix formation upon chain elongation. In the dimer and trimer (n = 2 and n = 3, respectively, in the structure reported above) the observed NOE connectivities are compatible with the 3(10)-helical arrangement, confirmed by the temperature coefficients of the amide proton resonances which suggest the presence of a hydrogen-bonded structure. The phi and psi dihedral angles of the structures obtained by molecular dynamics calculations are also compatible with the 3(10)-helix. Identification of the hydrogen-bond pattern indicate that C=O(i)- - -HN(i+3) hydrogen bonds, typical of the 3(10)-helical conformation, are highly probable in all low-energy structures. The CD spectra of these Aib-rich lactam-bridged oligopeptides, obtained in the same solvent system used for NMR experiments, provide important insight into the spectroscopic characteristics of the 3(10)-helix.

Anti-Bacterial Agents↗
PubMed2001

Folding study of an Aib-rich peptide in DMSO by molecular dynamics simulations.

To evaluate the ability of molecular dynamics (MD) simulations using atomic force-fields to correctly predict stable folded conformations of a peptide in solution, we show results from MD simulations of the reversible folding of an octapeptide rich in alpha-aminoisobutyric acid (2-amino-2-methyl-propanoic acid, Aib) solvated in di-methyl-sulfoxide (DMSO). This solvent generally prevents the formation of secondary structure, whereas Aib-rich peptides show a high propensity to form secondary structural elements, in particular 3(10)- and alpha-helical structures. Aib is, moreover, achiral, so that Aib-rich peptides can form left- or right-handed helices depending on the overall composition of the peptide, the temperature, and the solvation conditions. This makes the system an interesting case to study the ensembles of peptide conformations as a function of temperature by MD simulation. Simulations involving the folding and unfolding of the peptide were performed starting from two initial structures, a right-handed alpha-helical structure and an extended structure, at three temperatures, 298 K, 340 K, and 380 K, and the results are compared with experimental nuclear magnetic resonance (NMR) data measured at 298 K and 340 K. The simulations generally reproduce the available experimental nuclear Overhauser effect (NOE) data, even when a wide range of conformations is sampled at each temperature. The importance of adequate statistical sampling in order to reliably interpret the experimental data is discussed.

Dimethyl Sulfoxide↗
PubMed2001

Conformational study of an Aib-rich peptide in DMSO by NMR.

The strong propensity of 2-amino-2-methyl propanoic acid (Aib)-rich peptides to form stable helical structures is well documented. NMR analysis of the short peptide Z-(Aib)5-L-Leu-(Aib)2-OMe indicates the presence of a well-characterized 3(10)-helix even in dimethylsulfoxide (DMSO), a solvent known to disrupt helical structures. The structure remains stable at least up to 348 K. Stereospecific assignment of the diastereotopic methyls of Aib was achieved, with the assumption of a specific helical screw sense. The methyl more eclipsed with respect to the CO vector resonates at a higher field in the carbon dimension. Molecular dynamics simulations successfully predict the 3J(CHNH) coupling constant of Leu6 and most of the H-bonding pattern. Discrepancies were found for Aib3 and Aib7 amide protons which can be explained by a higher sensitivity of the simulations to the helix fraying at the end of the peptide and by the presence of extended conformations for Leu6 during most of the simulations.

Dimethyl Sulfoxide↗
PubMed2000

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrp) chimeric peptides.

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane-embedded G protein-coupled receptor (PTH1 Rc) present on the surface of cells in target tissues such as bone and kidney. This binding occurs in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34) PTH/PTHrP hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH in terms of bioactivity. The sites of incompatibility were identified at positions 5 in PTHrP and 19 in PTH. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two segmental hybrids: PTHrP(1-27)-[Tyr(34)]bPTH(28-34)-NH(2) (hybrid I) and PTHrP(1-18)-[Nal(23), Tyr(34)]bPTH(19-34)-NH(2) (hybrid II). Hybrid I is as active as PTH(1-34)NH(2) and more than two orders of magnitude more active than hybrid II. The conformational properties of the hybrids were studied in water/trifluoroethanol (TFE) mixtures and in aqueous solutions containing dodecylphosphocholine (DPC) micelles by CD, two-dimensional nmr and computer simulations. Upon addition of TFE to the aqueous solution, both hybrids undergo a coil-helix transition. The helix content in 1:1 water/TFE obtained by CD data is about 75% for both hybrids. In the presence of DPC, helix formation is observed at detergent concentrations above critical micellar concentration and the maximum helix content is of approximately 35 and approximately 30% for hybrid I and II, respectively. Combined nmr analysis, distance geometry, and molecular dynamics calculations suggest that, in both solvent systems, the biologically active hybrid I exhibits two flexible sites, centered at residues 12 and 19, connecting helical segments. The flexibility point at position 19 is not present in the poorly active hybrid II. Our findings support the hypothesis, proposed in our previous work, that in bioactive PTH analogues the presence and location of flexibility points between helical segments are essential for enabling them to fold into the bioactive conformation upon interaction with the PTH1 receptor.

Circular Dichroism↗
PubMed1999

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrP) point-mutated hybrids.

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34)PTH/PTHrP segmental hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH leading to substantial reduction in potency. The sites of incompatibility were identified as positions 5 in PTH and 19 in PTHrP. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two point-mutated PTH/PTHrP 1-34 hybrids in which the arginine residues at positions 19 and 21 of the native sequence of PTHrP have been replaced by valine (hybrid V(21)) and glutamic acid (hybrid E(19)), respectively, taken from the PTH sequence. Hybrid V(21) exhibits both high receptor affinity and biological potency, while hybrid E(19) binds weakly and is poorly active. The conformational properties of the two hybrids were studied in aqueous solution containing dodecylphosphocholine (DPC) micelles and in water/2,2, 2-trifluoroethanol (TFE) mixtures. Upon addition of TFE or DPC micelles to the aqueous solution, both hybrids undergo a coil-helix transition. The maximum helix content in 1 : 1 water/TFE, obtained by CD data for both hybrids, is approximately 80%. In the presence of DPC micelles, the maximum helix content is approximately 40%. The conformational properties of the two hybrids in the micellar system were further investigated by combined 2D-nmr, distance geometry (DG), and molecular dynamics (MD) calculations. The common structural motif, consisting of two helical segments located at N- and C-termini, was observed in both hybrids. However, the biologically potent hybrid V(21) exhibits two flexible sites, centered at residues 12 and 19 and connecting helical segments, while the flexibility sites in the weakly active hybrid E(19) are located at position 11 and in the sequence 20-26. Our findings support the hypothesis that the presence and location of flexibility points between helical segments are essential for enabling the active analogs to fold into the bioactive conformation upon interaction with the receptor.

Cell Line↗
PubMed1999

Design, synthesis, and conformational studies of the hGM-CSF derived peptide (13-27)-Gly-(75-87).

An analogue of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF), hGM-CSF(13-27)-Gly-(75-87) was synthesized by solid phase methodology. This analogue was designed to comprise helices A and C of the native growth factor, linked by a glycine bridge. Helices A and C form half of a four-helix bundle motif in the crystal structure of the native factor and are involved in the interaction with alpha- and beta-chains of the heterodimeric receptor. A conformational analysis of the synthetic analogue by CD, two-dimensional nmr spectroscopy, and molecular dynamics calculations is reported. The analogue is in a random structure in water and assumes a partially alpha-helical conformation in a 1 : 1 trifluoroethanol/water mixture. The helix content in this medium is approximately 70%. By 2D-nmr spectroscopy, two helical segments were identified in the sequences corresponding to helices A and C. In addition to medium- and short-range NOESY connectivities, a long-range cross peak was found between the Cbeta proton of Val(16) and NH proton of His(87) (using the numbering of the native protein). Experimentally derived interproton distances were used as restraints in molecular dynamics calculations, utilizing the x-ray coordinates as the initial structure. The final structure is characterized by two helical segments in close spatial proximity, connected by a loop region. This structure is similar to that of the corresponding domain in the x-ray structure of the native growth factor in which helices A and C are oriented in an antiparallel fashion. The N-terminal residues Gly-Pro of helix C are involved in an irregular turn connecting the two helical segments. As a consequence, helix C is appreciably shifted and slightly rotated with respect to helix A compared to the x-ray structure of the native growth factor. These small differences in the topology of the two helices could explain the lower biological activity of this analogue with respect to that of the native growth factor.

Drug Design↗
PubMed1999

Equilibrium unfolding CD studies of bovine beta-lactoglobulin and its 14-52 fragment at acidic pH.

Bovine beta-lactoglobulin represents an interesting example of context-dependent secondary structure induction. In fact, secondary structure predictions indicated that this beta-barrel protein has a surprisingly high alpha-helical preference, which was retained for short fragments. Cooperative transitions from the native beta-sheet to alpha-helical structures were additionally induced by organic solvents, in particular trifluoroethanol. As a result of this high alpha-helical preference, it has been proposed that non-native alpha-helical intermediates could be formed in the unfolding pathway of this protein. In order to provide a better understanding of the processes that underlie conformational plasticity in this protein, CD measurements in the presence of increasing amounts of urea and in the presence of organic solvents were performed. Urea unfolding studies, performed at pH 2.1 and 37 degrees C, revealed an apparent two-state transition, and afforded no evidence of non native alpha-helical intermediates. The protein treated with up to 6M urea, refolded to the native structure, while treatment with higher molar concentration urea, lead to partial misfolding. A 29-mer peptide covering the region of strands a and b of the intact protein, characterized by the presence of 4/3 heptad repeats, was synthesized and studied by CD in the presence of different solvents. On the basis of the obtained results, a mechanism was proposed to explain the structural transition from the beta to alpha structure, provoked by organic solvents in the intact protein.

Amino Acid Sequence↗
PubMed1999

Determination of the secondary structural elements of chicken liver fatty acid binding protein by two-dimensional homonuclear NMR.

A conformational study in solution of the fatty acid binding protein from chicken liver is presented. The nearly complete sequence-specific 1H resonance assignment was achieved from homonuclear two-dimensional nmr experiments using a sample of native protein. The principal elements of secondary structure were identified: 10 antiparallel beta-strands and one helical segment followed by a turn comprising 5 residues. These elements correspond closely with those of the crystal structure of the related protein, and two new secondary structural features obtained from the nmr data are the beta-sheet conformation between the first and the last beta-strand in the protein sequence, as well as a helical loop at the N-terminus of the polypeptide chain.

Amino Acid Sequence↗
PubMed1999

Conformational studies of a bicyclic, lactam-constrained parathyroid hormone-related protein-derived agonist.

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N- and C-termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1-34) analog [(Lys13-Asp17, Lys26-Asp30)]PTHrP(1-34)NH2, which contains lactam-constrained Lys-Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam-constrained analog to aqueous solution. By CD, 2D-NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13-18, and 26-31) with high flexibility around Gly12 and Arg19. Thus, the essential structural features observed in the aqueous-organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side-chain lactam constraints on the alpha-helical structure.

Amino Acid Sequence↗
PubMed1999

Characteristic MR lesion pattern and correlation of T1 and T2 lesion volume with neurologic and neuropsychological findings in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).

BACKGROUND AND PURPOSE: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an arteriopathy related to a genetic defect of the notch 3 gene on chromosome 19. The purpose of this study was to evaluate lesion distribution and volume using MR imaging and to correlate the lesion volume with the neurologic and neuropsychological findings. METHODS: Twenty members of two families (14 with CADASIL as determined by linkage analysis, six healthy) were studied with MR imaging. Two observers evaluated the MR findings semiquantitatively and quantitatively. MR results were then correlated with neurologic and neuropsychological findings. RESULTS: A typical pattern of lesion distribution in patients with CADASIL was found: the frontal lobe was the site with the highest lesion load, followed by the temporal lobe and the insula. The total lesion volume on T1-weighted MR images correlated significantly with the degree of disability and the degree of impairment in neuropsychological functions (including attention, memory, and conceptual and visuospatial functions). CONCLUSION: In CADASIL patients, a common pattern of cerebral lesion distribution is found. The total T1 lesion volume is an important parameter to correlate with disability, as it may prove to be helpful in predicting the natural history of the disease.

Adult↗
PubMed1998

Conformation and membrane activity of an analogue of the peptaibol antibiotic trichogin GA IV with a lipophilic amino acid at the N-terminus.

We have synthesized by solution-phase methods two analogues of the 11-residue lipopeptaibol antibiotic trichogin GA IV in which the N-terminal n-octanoyl group is replaced either by an N-acetylated 2-amino-2-methyl-L-undecanoic acid or by an N-acetylated alpha-aminoisobutyric acid. CD, FTIR absorption. and NMR analyses unequivocally show that the main structural features of trichogin GA IV are preserved in these analogues. Since only the peptide containing the lipophilic chain exhibits membrane-modifying properties, these results strongly support the view that moving the long acyl moiety from the Nalpha-blocking group to the side chain of the N-terminal extra-residue does not affect the conformational properties or the membrane activity of trichogin GA IV.

Amino Acid Sequence↗
PubMed1998

Solution conformational analysis of amphiphilic helical, synthetic analogs of the lipopeptaibol trichogin GA IV.

The step-by-step synthesis by solution methods of the [Ser2,5,6,9, Leu-OMe11] analog of trichogin GA IV is described. The four Ser residues have been incorporated into the sequence as replacements of the naturally occurring Gly residues to increase the amphiphilicity of the 3D-structure of the lipopeptaibol. A detailed solution conformational analysis has been performed on this undecapeptide and its prototypical [Leu-OMe11] trichogin GA IV analog using FTIR absorption and CD spectroscopies, and two-dimensional NMR under a variety of experimental conditions, including a membrane-mimetic environment. Both peptides adopt a mixed 3(10)/alpha-helical structure, which in the micellar system was found to be less flexible for the Ser-containing analog. For both analogs permeability measurements revealed membrane-modifying properties comparable to those of the natural lipopeptaibol.

Anti-Bacterial Agents↗