Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.

Robust stability of sequential multi-input multi-output quantitative feedback theory designs

This paper re-examines the stability of multi-input multi-output (MIMO) control systems designed using sequential MIMO quantitative feedback theory (QFT). In order to establish the results, recursive design equations for the SISO equivalent plants employed in a sequential MIMO QFT design are established. The equations apply to sequential MIMO QFT designs in both the direct plant domain, which employs the elements of plant in the design, and the inverse plant domain, which employs the elements of the plant inverse in the design. Stability theorems that employ necessary and sufficient conditions for robust closed-loop internal stability are developed for sequential MIMO QFT designs in both domains. The theorems and design equations facilitate less conservative designs and improved design transparency.

Scaling laws for the critical rupture thickness or common thin films

Despite decades of experimental and theoretical investigation on thin films, considerable uncertainty exists in the prediction of their critical rupture thickness. According to the spontaneous rupture mechanism, common thin films become unstable when capillary waves. at the interfaces begin to grow. In a horizontal film with symmetry at the midplane. unstable waves from adjacent interfaces grow towards the center of the film. As the film drains and becomes thinner, unstable waves osculate and cause the film to rupture, Uncertainty sterns from a number of sources including the theories used to predict film drainage and corrugation growth dynamics. In the early studies, (lie linear stability of small amplitude waves was investigated in the Context of the quasi-static approximation in which the dynamics of wave growth and film thinning are separated. The zeroth order wave growth equation of Vrij predicts faster wave growth rates than the first order equation derived by Sharma and Ruckenstein. It has been demonstrated in an accompanying paper that film drainage rates and times measured by numerous investigations are bounded by the predictions of the Reynolds equation and the more recent theory of Manev, Tsekov, and Radoev. Solutions to combinations of these equations yield simple scaling laws which should bound the critical rupture thickness of foam and emulsion films, In this paper, critical thickness measurements reported in the literature are compared to predictions from the bounding scaling equations and it is shown that the retarded Hamaker constants derived from approximate Lifshitz theory underestimate the critical thickness of foam and emulsion films, The non-retarded Hamaker constant more adequately bounds the critical thickness measurements over the entire range of film radii reported in the literature. This result reinforces observations made by other independent researchers that interfacial interactions in flexible liquid films are not adequately represented by the retarded Hamaker constant obtained from Lifshitz theory and that the interactions become significant at much greater separations than previously thought. (c) 2005 Elsevier B.V. All rights reserved.

On the chaotic rotation of a liquid-filled gyrostat via the Melnikov-Holmes-Marsden integral

The non-linear motions of a gyrostat with an axisymmetrical, fluid-filled cavity are investigated. The cavity is considered to be completely filled with an ideal incompressible liquid performing uniform rotational motion. Helmholtz theorem, Euler's angular momentum theorem and Poisson equations are used to develop the disturbed Hamiltonian equations of the motions of the liquid-filled gyrostat subjected to small perturbing moments. The equations are established in terms of a set of canonical variables comprised of Euler angles and the conjugate angular momenta in order to facilitate the application of the Melnikov-Holmes-Marsden (MHM) method to investigate homoclinic/heteroclinic transversal intersections. In such a way, a criterion for the onset of chaotic oscillations is formulated for liquid-filled gyrostats with ellipsoidal and torus-shaped cavities and the results are confirmed via numerical simulations. (c) 2006 Elsevier Ltd. All rights reserved.

Factors influencing the stability of cyclotides: Proteins with a circular backbone and cystine knot motif

Cyclotides are a large family of mini-proteins that have the distinguishing features of a head-to-tail cyclised backbone and a cystine knot formed by six conserved cysteine residues. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaceae families. The unique structural features of the cyclotides make them extremely resistant to chemical, thermal and proteolytic degradation. In this article we review recent Studies from our laboratory that dissect the role of the individual structural elements in defining the stability of cyclotides. The resistance of cyclotides to chemical and proteolytic degradation is in large part due to the cystine knot, whereas the thermal stability is I composite of several features including the cystine knot, the cyclic backbone and the hydrogen bonding network. A range of biological activities of cyclotides is critically dependent oil the presence of the cyclic backbone.

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.