Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.

On the forced matching numbers of bipartite graphs

Let G be a graph that admits a perfect matching. A forcing set for a perfect matching M of G is a subset S of M, such that S is contained in no other perfect matching of G. This notion has arisen in the study of finding resonance structures of a given molecule in chemistry. Similar concepts have been studied for block designs and graph colorings under the name defining set, and for Latin squares under the name critical set. There is some study of forcing sets of hexagonal systems in the context of chemistry, but only a few other classes of graphs have been considered. For the hypercubes Q(n), it turns out to be a very interesting notion which includes many challenging problems. In this paper we study the computational complexity of finding the forcing number of graphs, and we give some results on the possible values of forcing number for different matchings of the hypercube Q(n). Also we show an application to critical sets in back circulant Latin rectangles. (C) 2003 Elsevier B.V. All rights reserved.

The extended metamorphosis of a complete bipartite design into a cycle system

A K-t,K-t-design of order n is an edge-disjoint decomposition of K-n into copies of K-t,K-t. When t is odd, an extended metamorphosis of a K-t,K-t-design of order n into a 2t-cycle system of order n is obtained by taking (t - 1)/2 edge-disjoint cycles of length 2t from each K-t,K-t block, and rearranging all the remaining 1-factors in each K-t,K-t block into further 2t-cycles. The 'extended' refers to the fact that as many subgraphs isomorphic to a 2t-cycle as possible are removed from each K-t,K-t block, rather than merely one subgraph. In this paper an extended metamorphosis of a K-t,K-t-design of order congruent to 1 (mod 4t(2)) into a 2t-cycle system of the same order is given for all odd t > 3. A metamorphosis of a 2-fold K-t,K-t-design of any order congruent to 1 (mod 4t(2)) into a 2t-cycle system of the same order is also given, for all odd t > 3. (The case t = 3 appeared in Ars Combin. 64 (2002) 65-80.) When t is even, the graph K-t,K-t is easily seen to contain t/2 edge-disjoint cycles of length 2t, and so the metamorphosis in that case is straightforward. (C) 2004 Elsevier B.V. All rights reserved.

Diversity in the disulfide folding pathways of cystine knot peptides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif (CCK). This unique family was discovered only recently but contains over 50 known sequences to date. Various biological activities are associated with these peptides including antimicrobial and insecticidal activity. The knotted topology and cyclic nature of the cyclotides; poses interesting questions about the folding mechanisms and how the knotted arrangement of disulfide bonds is formed. Some studies have been performed on related inhibitor cystine knot (ICK) containing peptides, but little is known about the folding mechanisms of CCK molecules. We have examined the oxidative refolding and reductive unfolding of the prototypic member of the cyclotide family, kalata B1. Analysis of the rates of formation of the intermediates along the reductive unfolding pathway highlights the stability conferred by the cystine knot motif. Significant differences are observed between the folding of kalata B1 and an acyclic cystine knot protein, EETI-II, suggesting that the circular backbone has a significant influence in directing the folding pathway.

Metal Clips Induce Folding of a Short Unstructured Peptide into an a-Helix via Turn Conformations in Water. Kinetic versus Thermodynamic Products

Short peptides corresponding to two to four a-helical turns of proteins are not thermodynamically stable helices in water. Unstructured octapeptide Ac-His1*-Ala2-Ala3-His4*-His5*-Glu6-Leu7-His8*-NH2 (1) reacts with two [Pd ((NH2)-N-15(CH2)(2) (NH2)-N-15)(NO3)(2)] in water to form a kinetically stable intermediate, [{Pden}(2)-{(1,4)(5,8)-peptide}](2), in which two 19-membered metallocyclic rings stabilize two peptide turns. Slow subsequent folding to a thermodynamically more stable two-turn a-helix drives the equilibrium to [{Pden}(2)-{(1,5)(4,8)-peptide}] (3), featuring two 22-membered rings. This transformation from unstructured peptide via turns to an a-helix suggests that metal clips might be useful probes for investigating peptide folding.

Structure and folding of potato type II proteinase inhibitors: Circular permutation and intramolecular domain swapping

Potato type II serine proteinase inhibitors are proteins that consist of multiple sequence repeats, and exhibit a multidomain structure. The structural domains are circular permutations of the repeat sequence.. as a result or intramolecular domain swapping. Structural studies give indications for the origins of this folding behaviour, and the evolution of the inhibitor family.

Oxidative folding of the cystine knot motif in cyclotide proteins

The cyclotides are a large family of plant proteins that have a cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Despite the apparent complexity of their cystine knot motif it is possible to efficiently fold these proteins, as exemplified by oxidative folding studies on the prototypic cyclotide, kalata B1. This mini-review reports on the current understanding of the folding process in cyclotides. The synthesis and folding of these molecules paves the way for their application as stable molecular templates.

Protein disulfide isomerase: the structure of oxidative folding

Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI); by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell.

Kitchen servery to deck, Brannigan House, St Lucia, Brisbane

Corner of kitchen opens to deck via a series of folding windows.

Thermal effects in the evolution of initially layered mantle material

A simplified model for anisotropic mantle convection based on a novel class of rheologies, originally developed for folding instabilities in multilayered rock (MUHLHAUS et al., 2002), is extended ¨ through the introduction of a thermal anisotropy dependent on the local layering. To examine the effect of the thermal anisotropy on the evolution of mantle material, a parallel implementation of this model was undertaken using the Escript modelling toolkit and the Finley finite-element computational kernel (DAVIES et al., 2004). For the cases studied, there appears too little if any effect. For comparative purposes, the effects of anisotropic shear viscosity and the introduced thermal anisotropy are also presented. These results contribute to the characterization of viscous anisotropic mantle convection subject to variation in thermal conductivities and shear viscosities.