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Research demonstrates that analogues of human parathyroid hormone-1-34, each containing an insertion of a single beta-amino acid residue in positions 11,12, or 13, does not exhibit any structural disorder in this particular portion of the sequence of the hormone.

Examines the characteristics of human parathyroid hormone (PTH) and PTH-related protein. Biological properties of PTH; Stability of the N-terminal helix; Presence of amino acid residue in the helical structure of protein.

Human alpha-synuclein is a 140-amino acid protein of unknown function abundantly expressed in the brain and found in Lewy bodies, a characteristic feature of Parkinson's disease. Alpha-synuclein is random in water under physiological conditions, but the first approximately 100 residues interact with SDS micelles or acidic phospholipid small unilamellar vesicles and adopt an ordered conformation. The rest of the molecule remains disordered in the bulk of the solution. The conformation of the N-terminal portion of the molecule in lipids was described as an extended helix [Ramakrishnan, M., Jensen, P. H., and Marsh, D. (2003) Biochemistry 42, 12919-12926], as two distinct alpha-helices interrupted by a two-residue break [Chandra, S., Chen, X., Rizo, J., Jahn, R., and Sudhof, T. C. (2003) J. Biol. Chem. 278, 15313-15318], or as a noncanonical conformation, the alpha11/3 helix [Bussell, R., Jr., and Eliezer, D. (2003) J. Mol. Biol. 329, 763-778]. We characterized the topology of the different regions of alpha-synuclein relative to the surface of SDS micelles using spin probe-induced broadening of NMR signals, (15)N relaxation measurements, and fluorescence spectroscopy. Our results support the presence of two N-terminal helices, positioned on the surface of the micelle and separated by a flexible stretch. The region of residues 61-95 of the protein also adopts a helical conformation, but it is partially embedded in the micelle. These results could shed some light on the role of the membrane on the aggregation process of alpha-synuclein.

Pol peptide, an oligopeptide corresponding to the 27 C-terminal amino acids of DNA polymerase from herpes simplex virus type 1, has recently been suggested to translocate from endosomal compartments into the cytosol after being intracellularly delivered via a protein carrier. While an acidic environment was thought to be important for Pol peptide membrane translocation, the mechanism of translocation remains unclear. To investigate the influence of an acidic environment on the conformational properties of the peptide and on its propensity to interact with lipid bilayers, we characterized the structure of Pol peptide at different pH values by both circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The influence of detergent micelles, which mimic biological lipid membranes, on the peptide secondary structure was also studied. Our CD results indicate that the peptide is in a random conformation in aqueous solution at both acidic and basic pH, whereas in the presence of dodecylphosphocholine (DPC) micelles, it assumes a partial alpha-helical structure which is significantly pH-dependent. An NMR study confirmed that, in the presence of DPC micelles, a short C-terminal alpha-helix is present at pH 6.5, whereas almost two-thirds of the peptide (residues 10-26) fold into an extended amphipathic alpha-helix at pH 4.0. The orientation of Pol peptide relative to the DPC micelle was investigated using paramagnetic probes at both pH 4.0 and 6.5. These studies show that the peptide inserts deeply into the micelle at pH 4.0, whereas it is more exposed to the aqueous environment at pH 6.5. On the basis of these results, a model which might explain the mechanism of translocation of Pol peptide from acidic endosomes to the cytosol is discussed.

Parathyroid hormone-related protein (PTHrP) is expressed in a wide variety of cells where it acts as an autocrine and/or paracrine factor involved in regulation of cellular growth, differentiation, and embryonic development. It may also play a physiological endocrine role in calcium transport across the placenta or during lactation. The N-terminal portion, PTHrP-(1-34), retains all the calciotropic parathyroid hormone-like activity and is a lead structure for the design of novel, bone anabolic agents for the treatment of bone disorders such as osteoporosis. To characterize the putative bioactive conformation, we have carried out a detailed structural analysis of a series of three conformationally constrained PTHrP-(1-34)-based mono- and bicyclic lactam-containing biologically active analogs: (III) The conformational properties were studied by circular dichroisim, nuclear magnetic resonance spectroscopy, distance geometry calculations, and molecular dynamic simulations in water/trifluoroethanol (TFE) mixtures. The helical content in water of both monocyclic analogs I and II is approximately 22%; that of the bicyclic analog III is approximately 40%. In 30% TFE, all analogs reached a maximal helical content of 80%, corresponding to 26 or 27 residues out of 34 in a helical conformation. High-resolution structures obtained with 50:50 TFE/water revealed that all three analogs display two helical domains and a hinge region around Gly12-Lys13. The highly potent mono- and bicyclic agonists I and III display a second hinge around Arg19-Arg20 which is shifted to Ser14-Asp17 in the weakly potent monocyclic agonist II. We suggest that the presence and localization of discrete hinges in the sequence together with the high propensity for helicity of the C-terminal sequence and the enhancement of helical nucleation at the N-terminal sequence are essential for generating a PTH/PTHrP receptor-compatible bioactive conformation.

The conformation of the three cyclic antagonist analogs of parathyroid hormone-related protein (PTHrP)-(7-34) [[Lys13,Asp17]PTHrP-(7-34)NH2,[Lys26,Asp30 ]PTHrP-(7-34)NH2,[Lys13,Asp17,Lys26, Asp30]PTHrP-(7-34)NH2] is investigated by CD, NMR, and extensive computer simulations in aqueous solution and a TFE:water mixture. The structural analysis of these peptides, designed to stabilize different regions of the sequence in alpha-helical conformations, is an important step in addressing the correlation between helical content and binding affinity and bioactivity in this hormone-receptor system. Results from CD and NMR spectroscopy of all three analogues in aqueous solution indicate the presence of alpha-helix only in regions containing a 20-membered lactam ring. Upon addition of TFE, the three analogues display differences in the anticipated increase in helical content. The high-resolution structures produced at 50:50 TFE:water indicate specific differences in the extent and location of the helical regions. These conformations provide insight into the biological profiles of these analogues, reported in the previous manuscript [Bisello et al. (1997) Biochemistry 36, 3293-3299]. Since all three analogues are alpha-helical in the C-terminal region (residues 25-34 have been previously identified as containing the binding domain) and display similar binding affinities, we conclude that this conformational feature is important for the interaction between the peptide and the receptor. The extent of the helix (toward the N-terminus) and the presence of a hinge in the central region of the peptide play roles in the observed efficacy as measured by antagonism of PTH-stimulated adenylyl cyclase activity. The most active analogue consists of helical segments from residues 13-18 and 20-34, separated by a kink centered at Arg19.

This study is focused on the characterization of the interaction of the amphiphilic peptide bombolitin III (from the bumblebee Megabombus pennsylvanicus) with phospholipid monolayers and vesicles. It is shown that due to the amphiphilic character of its alpha-helical conformation this water-soluble peptide is able to interact in an ordered fashion with phospholipid organized structures. Depending on the temperature, the subphase, and the particular phosphatidylcholine used, the mixed peptide-phospholipid monolayers can be homogeneous or display phase separation. This behavior was observed by means of the Langmuir film balance technique, coupled with an epifluorescence microscope. In well-defined conditions it is possible to visualize the formation of phase-separated peptide domains at the air-water interface and to study the effect of their presence on the organization of the lipid. The action of phospholipase A2 at the lipid-peptide interface was also followed by means of fluorescence microscopy: some evidence that the enzyme preferentially hydrolyzes the phospholipid that is in contact with the peptide is presented. Furthermore, the presence of bombolitin III in L-alpha-DLPC monolayers causes an increase in the initial speed of degradation with phospholipase A2. These results are in agreement with previous findings that show that the bombolitins are activators in vitro of phospholipase A2. Experiments were also performed with peptide fragments corresponding to the alpha-helical sequences of the protein uteroglobin: despite some amphiphilic character, these peptides do not interact strongly with phospholipid monolayers. Only one of these peptides (corresponding to the helix 4-14 in uteroglobin) is adsorbed in the monolayer in a similar fashion to bombolitin III but does not cause an increase in the activity of phospholipase A2.

The heptadecapeptides bombolitin I and bombolitin III are two of a series of peptides postulated to be biologically active within a membrane environment. In the preceding paper [Bairaktari, E., Mierke, D.F., Mammi, S., & Peggion, E. (1990) Biochemistry (preceding paper in this issue)] the conformational preferences of these peptides in the presence of SDS surfactant micelles, a mimetic for biological membranes, were examined. During these studies the conformations of these peptides were investigated in aqueous solutions by circular dichroism and nuclear magnetic resonance. A large difference was observed for the two peptides. Bombolitin I lacks any observable secondary structure in aqueous solution, independent of temperature, pH, and concentration. In striking contrast, bombolitin III adopts a well-defined alpha-helix at concentrations greater than 1.3 mM. This is indeed surprising given the great similarity of the two peptides. The alpha-helix of bombolitin III is pH dependent, with a great decrease in the observed secondary structure at pH values below 3.5. This observation could only be due to the protonation of the Asp residue at the fifth position. These findings suggest that the secondary structure arises from molecular aggregation of bombolitin III through the formation of a salt bridge involving the Asp side chain. The alpha-helix observed at "high" concentration (greater than 2.5 mM) has been characterized by CD and by the NOE's measured throughout a majority of the peptide. The experimentally determined structure has been energy refined with restrained molecular dynamics. The conformational results from this study are then compared with the conformations found in the presence of surfactant micelles.

The heptadecapeptides bombolitin I and bombolitin III are two members of a series of biologically active peptides postulated to be membrane active. In order to understand the effects of the membrane on the secondary structure of the peptides, we have carried out the conformational characterization of bombolitins I and III in the presence of SDS micelles using circular dichroism, nuclear magnetic resonance, and computer simulations. The characteristic bands in the circular dichroism spectra indicate an alpha-helix content of approximately 60% in bombolitin III and 70% in bombolitin I. The observation of NOE's quite distinctive for such secondary structure strongly supports the CD results. The conformational preferences of the two bombolitins derived from CD and NMR were then energetically refined with molecular dynamics simulations. The results from the spectroscopic examination were utilized as input for the simulations, the CD results for generation of the initial structure, and the NOE's as constraints during the simulations. The results from the different techniques employed are in complete agreement.

Human little gastrin is a 17 amino acid peptide that adopts a random conformation in water and an ordered structure in sodium dodecyl sulfate (SDS) micelles as well as in trifluoroethanol (TFE). The circular dichroism spectra in these two media have the same shape, indicative of a similar preferred conformation [Mammi, S., Mammi, N. J., Foffani, M. T., Peggion, E., Moroder, L., & Wünsch, E. (1987) Biopolymers 26, S1-S10]. We describe here the assignment of the proton NMR resonances and the conformational analysis of [Ahx15]little gastrin in SDS micelles. Two-dimensional correlation techniques form the basis for the assignment. The conformational analysis utilized NOE's, NH to C alpha H coupling constants, and the temperature coefficients of the amide chemical shifts. The NMR data indicate a helical structure in the N-terminal portion of the peptide. These results are compared with the conformation that we recently proposed for a minigastrin analogue (fragment 5-17 of [Ahx15]little gastrin) in TFE.

Synthesis, conformation, and biological properties of the Ala10 analogue of des-Trp1,Nle12-minigastrin are reported. Replacement of the Gly residue in the original sequence with Ala remarkably changes the conformational preference of the hormone in trifluoroethanol. CD and NMR results indicate that the conformational change is mainly located in the C-terminal portion of the molecule, with probable extension of the N-terminal alpha-helix throughout the entire sequence. The structural modification causes a 10-fold decrease in the biological potency of the hormone, which is about as active as the C-terminal tetrapeptide amide. These findings support our previous hypothesis that the optimal bioactive conformation of the native hormone is U-shaped, with mutual interactions among the two end segments.

The 1H NMR spectrum of the title peptide, H-Leu-(Glu)5-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2, in 90% H2O/10% D2O was assigned by two-dimensional methods, and the displacement of the proton resonances upon addition of 2,2,2-trifluoroethanol (TFE) was followed. This permitted the assignment of the spectrum in 90% TFE/10% D2O. While the water conformation of the minigastrin analogue is random, the CD spectrum indicates that an ordered structure is present in TFE. Variable-temperature NMR data in this medium show that six amide protons have low temperature coefficients, two of the five Glu's, Trp, Nle, Asp, and Phe. These results were interpreted in terms of an alpha-helical stretch comprising the Leu and the five Glu residues and a 3(10)-helix initiated by a beta-turn at the sequence -Ala-Tyr-Gly-Trp-. Both CD and NMR data at different solvent compositions show two regions of conformational change, between 20 and 25% water and above 60% water.