Different period gene repeats take 'turns' at fine-tuning the circadian clock.

The repetitive region of the circadian clock gene period in Drosophila pseudoobscura consists predominantly of a pentapeptide sequence whose consensus is NSGAD. In D. melanogaster, this region is replaced by a dipeptide Thr-Gly repeat, which plays a role in the thermal stability of the circadian phenotype. The Thr-Gly repeat has been shown to form a type II or III beta-turn, whose conformational monomer is (Thr-Gly)3. Here we report, using conformational analyses, that both an NSGAD pentapeptide, and a polymer of the same sequence, form type II beta-turns. Thus two peptide sequences, whose amino-acid composition is very different, nevertheless form the same secondary structure. The implications of these structures for clock function are discussed.

Conformational study of the Thr-Gly repeat in the Drosophila clock protein, PERIOD.

Recent results with the Drosophila melanogaster period gene suggest that the apparently conserved repetitive motif (Thr-Gly)n encoded by this gene may play an important role in the temperature compensation of the circadian clock. We have therefore initiated both a theoretical and experimental conformational analysis of (Thr-Gly)n peptides. By using a build-up method, it is clear that the hexapeptide (Thr-Gly)3 represents a 'conformational monomer' and generates a stable type II or type III beta-turn. Circular dichroism and nuclear magnetic resonance spectra of synthetic (Thr-Gly)3 and poly(Thr-Gly) peptides revealed that these peptides exhibit flexible conformations, especially in more polar environments and at higher temperatures. We speculate that this flexibility may illuminate our understanding of both the molecular mechanism of temperature compensation and the systematic geographical distribution within Europe of the Thr-Gly length polymorphism in D. melanogaster.

Solid-state studies on synthetic fragments and analogues of elastin.

A series of synthetic fragments and analogues of elastin have been investigated, in the solid state, by means of differential scanning calorimetry and thermally stimulated current. Most of the polypeptides were shown to possess both amorphous regions and segments of long-range order. Water, which interacts preferentially with the amorphous zones, behaves as plasticizer, i.e. facilitates the localized motions of polypeptide chains. The results obtained have been correlated with elastin elasticity, in particular as far as the fundamental destructuring role of water is concerned.

Synthesis and structural studies of a pentapeptide sequence of elastin. Poly (Val-Gly-Gly-Leu-Gly).

Poly (Val-Gly-Gly-Leu-Gly), a polypeptide mimicking the physico-chemical properties of the glycine-rich regions of elastin, has been synthesized and studied both in solution and in the aggregated state. By comparison, also the conformation of different "monomeric" units has been investigated. The polymer showed increased disorder with respect to the "monomers", the molecular conformation being accounted for by a more or less random collection of isolated beta-turns. Nevertheless, in the solid state the polymer is able to adopt supramolecular structures reminiscent of those found for elastin.

Phase transitions and chain dynamics, in the solid state, of a pentapeptide sequence of elastins.

Differential scanning calorimetry (d.s.c.) and thermally stimulated current (t.s.c.) have been applied to the study of thermal transitions and dielectric relaxations of a pentapeptide sequence: Gly-Leu-Gly-Gly-Val of elastin. The manifestation of the glass transition has been observed by both techniques. The analysis of the fine structure of t.s.c. spectra reveals the existence of local order in the amorphous phase upon physical ageing. In the 'true' amorphous phase, cooperative motions of sequences of various length are observed. The corresponding activation parameters are characteristic of the 'structure' of the amorphous phase and might be used as reference for further studies.

Molecular dynamics study of the conformational behavior of a representative elastin building block: Boc-Gly-Val-Gly-Gly-Leu-OMe.

The conformational behavior of the synthetic peptide, Boc-Gly-Val-Gly-Gly-Leu-OMe, containing the X-Gly-Gly and Gly-Gly-X (X = Val or Leu) repeating sequences and constituting a fragment of elastin was investigated by molecular mechanics and molecular dynamics (MD) simulation. The results suggest that, irrespective of the approximations used, the molecule shows a manifold of low energy conformations characterized by gamma-turns and type II beta-turns. Furthermore, MD simulations point out a conformational floppiness due to very low barriers between different conformations. Experimental CD measurements in a virtually apolar medium (dioxane--epsilon = 2.209), which better mimics the vacuum conditions of the simulation, support the theoretical results. The general emerging picture, indicating the molecule as characterized by a combination of flexibility with conformational preferences, is in agreement with previous experimental findings and enriches of new aspects the description of the microscopic behavior of this molecule suggesting more detailed interpretation of previous data.

Polypeptide models of elastin: CD and NMR studies on synthetic poly(X-Gly-Gly).

Poly(X-Gly-Gly), simple structural models for the hydrophobic, proline-devoid, regions of elastin, have been synthesized and studied by circular dichroism and NMR spectroscopies. The results gave evidence of type II beta-turns as the only ordered structure present in the polymers. The stability of the turns has been shown to decrease on hydration and to increase in the series Leu less than Ala less than Val less than Ile.

Spectroscopic studies on elastin-like synthetic polypeptides.

Spectroscopic studies on synthetic polypeptides containing the unit-X-G-G (X=V or L) are reported. The sequences, constituting either fragments or model of elastin, were shown to adopt type II beta-turns together with an ensemble of unordered conformations. Furthermore, it was found that the stability of the beta-turns was depending on the nature of the X residue, on the hydration of the chain and, in the case of the sequence G-V-G-G-L, was decreasing by increasing the length of the chain.

Synthetic fragments and analogues of elastin. I. The synthesis.

The synthesis of some repetitive sequences of elastin and their simplified analogues, all comprising the structural unit Gly-X-Gly (X = Val, Leu, Ala), is described. In particular, the following peptides and polypeptides were synthesized and characterized: Boc-Gly-Val-Gly-Gly-Leu-OMe, Boc-Gly-Leu-Gly-Gly-Val-OMe, Boc-(Gly-Val-Gly-Gly-Leu)2-OMe, Boc-(Gly-Val-Gly-Gly-Leu)3-OMe, Boc-Gly-Val-Gly-Gly-OEt, Boc-Leu-Gly-Gly-Leu-OMe, Boc-Val-Gly-Gly-Val-OMe, poly(Ala-Gly-Gly), poly(Val-Gly-Gly), and poly(Leu-Gly-Gly). In every case, the synthesis was accomplished by classical procedures in solution, by using the p-nitrophenyl ester method for the polycondensation step, and the mixed anhydride or the azide methods for the coupling steps.

Synthetic fragments and analogues of elastin. II. Conformational studies.

Conformational studies on synthetic repetitive sequences and analogues of elastin are described. CD and nmr measurements gave evidence of flexible beta-turns as the dominant structural feature whose stability was found to decrease by increasing the number of repetitive units. The sequences comprised the structural unit Gly-X-Gly (X = Val, Leu, Ala), with X-Gly or Gly-Gly located at the corners of the bend. Based on that, it is proposed that these regions of elastin, unlike the proline-containing sequences, contribute to the elasticity of the protein through a classical mechanism in terms of the rotational isomeric state theory.

Electron microscopic evidence for elastin-like supramolecular organization in synthetic polytripeptides.

Electron microscope studies have been carried out on polytripeptides comprising the sequences -Pro-X-Gly- and -X-Pro-Gly- (X = Val, Ile, Met). These polymers have previously been shown to either contain or lack secondary structure. The formation of aligned filaments and also, in some cases, of banded fibers has been demonstrated. Independent of predicted solution conformation the supramolecular structures appeared to be very similar to those previously demonstrated for elastin and its soluble derivatives. The possibility that supramolecular organization is not a necessary consequence of molecular ordered structures is put forward and discussed. With respect to the molecular structure of elastin, the results suggest caution in the interpretation of electron microscope observation at the molecular scale.

Conformational studies on polypeptide models of collagen. Poly(Gly-Pro-Val), poly(Gly-Pro-Met), poly(Gly-Val-Pro) and poly(Gly-Met-Pro).

The title polytripeptides were synthesized and studied experimentally, by circular dichroism, and theoretically, by quantum mechanical methods. With the exception of poly(Gly-Pro-Val), which was found to be essentially structureless in solution, the other polymers adopt folded conformations, most probably of type II beta-bends. Conclusions from theoretical studies were generally in agreement with the experimental results. In particular, it is noteworthy that the optimized (phi, psi) maps for poly(Gly-Pro-Met) showed the absolute minimum (phi = 60 degree, psi = 0 degrees) located inside the beta II bend space.

Experimental and theoretical conformational studies on polypeptide models of collagen. Poly(Gly-Pro-Ile) and poly(Gly-Ile-Pro).

The synthetic polytripeptides poly(Gly-Pro-Ile) and poly(Gly-Ile-Pro) were studied both experimentally (mainly by circular dichroism spectroscopy) and theoretically by quantum mechanical methods. Poly(Gly-Ile-Pro) adopts a collagen-like structure under favourable conditions while the isomeric poly(Gly-Pro-Ile) does not. Theoretical studies emphasize severe intrastrand interactions which limit the main chain conformations in the case of poly(Gly-Ile-Pro). On the other hand, both the side cahin and the backbone in poly(Gly-Pro-Ile) can take up many different local conformations. Therefore, it seems that the conformational behaviour of synthetic polytripeptides can be at least partially explained in terms of local interactions.

Interactions of human and bovine elastins with lipids: their proteolysis by elastase.

The elastase-catalyzed hydrolysis of elastin extracted from young and old human aortas was compared with that of elastin from a bovine source. The course of the elastolysis was followed in the presence of sodium taurocholate and some unsaturated fatty acids. The results obtained suggest interesting correlations between the effects of aging on elastin, interaction of elastin with lipids and its susceptibility to proteolysis by elastase.

Concentration-dependent conformational transition of alpha-elastin in aqueous solution.

Circular dichroism measurements on aqueous solutions of alpha-elastin have given evidence of beta-bend structure at elevated concentrations. When Ca2+ was added, the concentration-dependent conformational transition was somewhat inhibited and the binding of the metal ion was shown, by means of equilibrium dialysis, to be essentially independent from alpha-elastin concentration in the range of 1 to 10 mg/ml. The results obtained are discussed in connection with the elasticity and calcification of elastin.

Conformational transitions of alpha-elastin.

The alpha-elastin, the soluble product obtained from elastin by oxalic acid hydrolysis, has been studied from a conformational point of view in different conditions using circular dichroism spectroscopy. The spectral variations of the alpha-elastin dissolved in water and in different organic solvents/water mixtures have been examined in order to establish the effect of the solvents on the structural properties of the protein. The effect of calcium ions has also been studied on alpha-elastin dissolved either in water or in organic solvents. Finally, the effect of pH and temperature on the conformational properties of the alpha-elastin has been investigated.

Interaction of Pt(II) complexes with DNAs from various sources. A circular dichroism study.

The interaction of cis- and trans-Pt(NH3)2Cl2 with DNAs of different base composition was studied by means of circular dichroism (CD) spectroscopy. The spectra obtained indicated a partial transformation of the secondary structure of DNA (B- less than C transition) with an increased helix winding. Subtle differences were noted between the two isomers, possibly related to changes in base orientation due to the different molecular geometries of the P+(II) complexes.