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.

Discovery, structure and biological activities of the cyclotides

The cyclotides are a family of small disulfide rich proteins that have a cyclic peptide backbone and a cystine knot formed by three conserved disulfide bonds. The combination of these two structural motifs contributes to the exceptional chemical, thermal and enzymatic stability of the cyclotides, which retain bioactivity after boiling. They were initially discovered based on native medicine or screening studies associated with some of their various activities, which include uterotonic action, anti-HIV activity, neurotensin antagonism, and cytotoxicity. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaccae families and their natural function in plants appears to be in host defense: they have potent activity against certain insect pests and they also have antimicrobial activity. There are currently around 50 published sequences of cyclotides and their rate of discovery has been increasing over recent years. Ultimately the family may comprise thousands of members. This article describes the background to the discovery of the cyclotides, their structural characterization, chemical synthesis, genetic origin, biological activities and potential applications in the pharmaceutical and agricultural industries. Their unique topological features make them interesting from a protein folding perspective. Because of their highly stable peptide framework they might make useful templates in drug design programs, and their insecticidal activity opens the possibility of applications in crop protection.

The evaluation of the spatial and temporal stability of sugarcane farm performance based on yield and commercial cane sugar

In broader catchment scale investigations, there is a need to understand and ultimately exploit the spatial variation of agricultural crops for an improved economic return. In many instances, this spatial variation is temporally unstable and may be different for various crop attributes and crop species. In the Australian sugar industry, the opportunity arose to evaluate the performance of 231 farms in the Tully Mill area in far north Queensland using production information on cane yield (t/ha) and CCS ( a fresh weight measure of sucrose content in the cane) accumulated over a 12-year period. Such an arrangement of data can be expressed as a 3-way array where a farm x attribute x year matrix can be evaluated and interactions considered. Two multivariate techniques, the 3-way mixture method of clustering and the 3-mode principal component analysis, were employed to identify meaningful relationships between farms that performed similarly for both cane yield and CCS. In this context, farm has a spatial component and the aim of this analysis was to determine if systematic patterns in farm performance expressed by cane yield and CCS persisted over time. There was no spatial relationship between cane yield and CCS. However, the analysis revealed that the relationship between farms was remarkably stable from one year to the next for both attributes and there was some spatial aggregation of farm performance in parts of the mill area. This finding is important, since temporally consistent spatial variation may be exploited to improve regional production. Alternatively, the putative causes of the spatial variation may be explored to enhance the understanding of sugarcane production in the wet tropics of Australia.

Determination of thermal and optical parameters of melanins by photopyroelectric spectroscopy

Photopyroelectric spectroscopy (PPE) was used to study the thermal and optical properties of melanins. The photopyroelectric intensity signal and its phase were independently measured as a function of wavelength and chopping frequency for a given wavelength in the saturation part of the PPE spectrum. Equations for both the intensity and the phase of the PPE signal were used to fit the experimental results. From these fits we obtained for the first time, with great accuracy, the thermal diffusivity coefficient, the thermal conductivity, and the specific heat of the samples, as well as a value for the condensed phase optical gap, which we found to be 1.70 eV. (c) 2005 American Institute of Physics.

Thermal and electrical currents in nanoscale electronic interferometers

We theoretically study thermal transport in an electronic interferometer comprising a parallel circuit of two quantum dots, each of which has a tunable single electronic state which are connected to two leads at different temperature. As a result of quantum interference, the heat current through one of the dots is in the opposite direction to the temperature gradient. An excess heat current flows through the other dot. Although locally, heat flows from cold to hot, globally the second law of thermodynamics is not violated because the entropy current associated with heat transfer through the whole device is still positive. The temperature gradient also induces a circulating electrical current, which makes the interferometer magnetically polarized.

ATM signaling and genornic stability in response to DNA damage

DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangietasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed. (C) 2004 Published by Elsevier B.V.

The relationship between knee strength and functional stability before and after anterior cruciate ligament reconstruction

Functional stability of the knee is dependent on an intact ligamentous system and the timely and efficient contraction of supporting musculature. The aim of this study was to assess the relationship between muscle strength and functional stability in 31 patients pre- and post-operatively, following a unilateral anterior cruciate ligament rupture. All subjects underwent reconstructive surgery using semitendonosis and gracilis tendons. Isokinetic strength assessment of quadriceps and hamstring muscles was performed at a rate of movement of 60% and 120degrees/s. Functional stability was determined by performance during five functional stability tests that included the shuttle run, side step, carioca, single and triple hop tests. Pearson's correlation coefficient statistics were applied to pre-operative and post-operative data respectively. These analyses demonstrated a significant positive correlation between quadriceps strength indices at both testing speeds and the two hop tests pre-operatively (p's < 0.007) and between quadriceps strength indices at both speeds and all five functional tests post-operatively (p's < 0.01). Assessed using Steiger's formula, there was a significant increase in the correlation between quadriceps strength indices and three functional tests post-operatively compared to pre-operatively (p < 0.05). No significant correlation between hamstring strength indices and functional scores existed pre- or post-operatively. This study has shown a significant correlation exists between quadriceps strength indices and functional stability both before and after surgery, this relationship does not reach significance between hamstring strength indices and functional stability. (C) 2002 Orthopaedic Research Society. Published by Elsevier Science 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.

Isomeric distribution and catalyzed isomerization of cobalt(III) complexes with pentadentate macrocyclic ligands. Importance of hydrogen bonding

We have investigated the isomeric distribution and rearrangement of complexes of the type [CoXLn](2+,3+) (where X = Cl-, OH-, H2O, and L-n represents a pentadentate 13-, 14-, and 15-membered tetraaza or diaza-dithia (N-4 or N2S2) macrocycle bearing a pendant primary amine). The preparative procedures for chloro complexes produced almost exclusively kinetically preferred cis isomers (where the pendant primary amine is cis to the chloro ligand) that can be separated by careful cation-exchange chromatography. For L-13 and L-14 the so-called cis-V isomer is isolated as the kinetic product, and for L-15 the cis-VI form (an N-based diastereomer) is the preferred, while for the L-14(S) complex both cis-V and trans-I forms are obtained. All these complexes rearrange to form stable trans isomers in which the pendent primary amine is trans to the monodentate aqua or hydroxo ligand, depending on pH and the workup procedure. In total 11 different complexes have been studied. From these, two different trans isomers of [CoCIL14S](2+) have been characterized crystallographically for the first time in addition to a new structure of cis-V-[CoCIL14S](2+); all were isolated as their chloride perchlorate salts. Two additional isomers have been identified and characterized by NMR as reaction intermediates. The remaining seven forms correspond to the complexes already known, produced in preparative procedures. The kinetic, thermal, and baric activation parameters for all the isomerization reactions have been determined and involve large activation enthalpies and positive volumes of activation. Activation entropies indicate a very important degree of hydrogen bonding in the reactivity of the complexes, confirmed by density functional theory studies on the stability of the different isomeric forms. The isomerization processes are not simple and even some unstable intermediates have been detected and characterized as part of the above-mentioned 11 forms of the complexes. A common reaction mechanism for the isomerization reactions has been proposed for all the complexes derived from the observed kinetic and solution behavior.