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.

Reducing the height of a circular array of monopoles using top hats and dielectric coatings

Using a Radial Guide Field Matching Method, an investigation is performed into reducing the height of an electronically steered circular array of monopole antennas composed of a central active element surrounded by passive elements being either short- or open-circuited. It is shown that a considerable height reduction can be achieved using top hats attached to monopoles ends and by applying dielectric coating underneath the top hats. The trade-off in achieving height reduction is narrower impedance bandwidth.

Effect of mutual coupling on the interference rejection capabilities of linear and circular arrays in CDMA systems

This paper is concerned with assessing the interference rejection capabilities of linear and circular array of dipoles that can be part of a base station of a code-division multiple-access cellular communication system. The performance criteria for signal-to-interference ratio (SIR) improvement employed in this paper is the spatial interference suppression coefficient. We first derive an expression for this figure of merit and then analyze and compare the SIR performance of the two types of arrays. For a linear array, we quantitatively assess the degradation in SIR performance, as we move from array broadside to array end-fire direction. In addition, the effect of mutual coupling is taken into account.

A low-profile dual-band antenna for a wireless LAN access point

The design of dual-band 2.45/5.2 GHz antenna for an acces point of a Wireless Local Area Network (LAN) is presented. The proposed antenna is formed by a Radial Line Slot Array (RLSA) operating at 2.4 GHz and a Microstrip patch working at 5.2 GHz, both featuring circular polarization. The design of this antenna system is accomplished using commercially available Finite Element software. High Frequency Structure Simulator (HFSS) of Ansoft and an in-house developed iteration procedure. The performance of the designed antenna is assessed in terms of return loss (RL), radiation pattern and polarization purity in the two frequency bands.

Winding patterns for biplanar MRI shim coils with rectangular and circular target-field regions

A method is presented for calculating the winding patterns required to design independent zonal and tesseral biplanar shim coils for magnetic resonance imaging. Streamline, target-field, Fourier integral and Fourier series methods are utilized. For both Fourier-based methods, the desired target field is specified on the surface of the conducting plates. For the Fourier series method it is possible to specify the target field at additional depths interior to the two conducting plates. The conducting plates are confined symmetrically in the xy plane with dimensions 2a x 2b, and are separated by 2d in the z direction. The specification of the target field is symmetric for the Fourier integral method, but can be over some asymmetric portion pa < x < qa and sb < y < tb of the coil dimensions (-1 < p < q < 1 and -1 < s < t < 1) for the Fourier series method. Arbitrary functions are used in the outer sections to ensure continuity of the magnetic field across the entire coil face. For the Fourier series case, the entire field is periodically extended as double half-range sine or cosine series. The resultant Fourier coefficients are substituted into the Fourier series and integral expressions for the internal and external magnetic fields, and stream functions on both the conducting surfaces. A contour plot of the stream function directly gives the required coil winding patterns. Spherical harmonic analysis of field calculations from a ZX shim coil indicates that example designs and theory are well matched.

Extremal circular permutations with concave functions

Let {a(1), a(2), ..., a(n)} be a set of n distinct real numbers and let alpha(1), alpha(2), ..., alpha(n) an be a permutation of the numbers. We construct the permutation to maximise L-f = Sigma(i=1)(n) f(\alpha(i+1) - alpha(i)\), for any increasing concave function f, where we denote alpha(n+1) equivalent to alpha(1). The optimal permutation depends on the particular numbers {a(1), a(2), ..., a(n)} and the function f, contrary to a postulate by Chao and Liang (European J. Combin. 13 (1992) 325). (C) 2004 Elsevier Ltd. All rights reserved.

Transient bending of a piezoelectric circular plate

Thin, piezoelectric circular plates are frequently used as active components in transducer and smart materials applications. This paper reports on the exact, explicit solution for the transient motion of a piezoelectric circular plate, built-in or simply supported on the edge and electrically grounded over the entire surface. Expressed by elementary Bessel functions and obtained via exact inverse Laplace transforms, the solution enables the efficient calculation of accurate system parameters. (C) 2004 Elsevier Ltd. All rights reserved.

Investigation of in vitro transdermal absorption of fentanyl from patches placed on skin samples obtained from various anatomic regions of dogs

Objective-To investigate in vitro transdermal absorption of fentanyl from patches through skin samples obtained from various anatomic regions of dogs. Sample Population-Skin samples from 5 Greyhounds. Procedure-Skin samples from the dogs' thoracic, neck, and groin regions were collected postmortem and frozen. After samples were thawed, circular sections were cut and placed in Franz-type diffusion cells in a water bath (32degreesC). A commercial fentanyl patch, attached to an acetate strip with a circular hole, was applied to each skin sample. Cellulose strips were used as control membranes. Samples of receptor fluid in the diffusion cells were collected at intervals for 48 hours, and fentanyl concentrations were analyzed by use of high-performance liquid chromatography. Results-Mean +/- SD release rate of fentanyl from the patch, defined by its absorption rate through the non-rate-limiting cellulose membrane, was linear during the first 8 hours (2.01 +/- 0.05 pg/cm(2) of cellulose membrane/h) and then decreased. Fentanyl passed through skin from the groin region at a faster rate and with a significantly shorter lag time, compared with findings in neck or thoracic skin samples. Conclusions and Clinical Relevance-In vitro, fentanyl from a patch was absorbed more quickly and to a greater extent through skin collected from the groin region of dogs, compared with skin samples from the thoracic and neck regions. Placement of fentanyl patches in the groin region of dogs may decrease the lag time to achieve analgesia perioperatively; however, in vivo studies are necessary to confirm these findings.

Conserved structural and sequence elements implicated in the processing of gene-encoded circular proteins

The cyclotides are the largest family of naturally occurring circular proteins. The mechanism by which the termini of these gene-encoded proteins are linked seamlessly with a peptide bond to form a circular backbone is unknown. Here we report cyclotide-encoding cDNA sequences from the plant Viola odorata and compare them with those from an evolutionarily distinct species, Oldenlandia affinis. Individual members of this multigene family encode one to three mature cyclotide domains. These domains are preceded by N-terminal repeat regions (NTRs) that are conserved within a plant species but not between species. We have structurally characterized peptides corresponding to these NTRs and show that, despite them having no sequence homology, they form a structurally conserved alpha-helical motif. This structural conservation suggests a vital role for the NTR in the in vivo folding, processing, or detoxification of cyclotide domains from the precursor protein.

Capped acyclic permutants of the circular protein kalata B1

The cyclotides are a family of head-to-tail cyclized peptides that display exceptionally high stability and a range of biological activities. Acyclic permutants that contain a break in the circular backbone have been reported to be devoid of the haemolytic activity of the prototypic cyclotide kalata B1, but the potential role of the charges at the introduced termini in this loss of membraneolytic activity has not been fully determined. In this study, acyclic permutants of kalata B1 with capped N- and G termini were synthesized and found to adopt a native fold. These variants were observed to cause no measurable lysis of erythrocytes, strengthening the connection between backbone cyclization and haemolytic activity. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Structure-function studies of the plant cyclotides: The role of a circular protein backbone

The traditional idea of proteins as linear chains of amino acids is being challenged with the discovery of miniproteins that contain a circular backbone. The cyclotide family is the largest group of circular proteins and is characterized by an amide-circularized protein backbone and six conserved cysteine residues. These conserved cysteines are paired to form a knotted network of disulfide bonds. The combination of the circular backbone and a cystine knot, known as the cyclic cystine knot (CCK) motif, confers exceptional stability upon the cyclotides. This review discusses the role of the circular backbone based on studies of both the oxidative folding of kalata B1, the prototypical cyclotide, and a comparison of the structure and activity of kalata B1 and its acyclic permutants.

Patch and whole of surface mass-transfer rate measurements on a stepped rotating cylinder electrode

A recently developed whole of surface electroplating technique was used to obtain mass-transfer rates in the separated flow region of a stepped rotating cylinder electrode. These data are compared with previously reported mass-transfer rates obtained with a patch electrode. It was found that the two methods yield different results, where at lower Reynolds numbers, the mass-transfer rate enhancement was noticeably higher for the whole of the surface electrode than for the patch electrode. The location of the peak mass transfer behind the step, as measured with a patch electrode, was reported to be independent of the Reynolds number in previous studies, whereas the whole of the surface electrode shows a definite Reynolds number dependence. Large eddy simulation results for the recirculating region behind a step are used in this work to show that this difference in behavior is related to the existence of a much thinner fluid layer at the wall for which the velocity is a linear junction of distance from the wall. Consequently, the diffusion layer no longer lies well within a laminar sublayer. It is concluded that the patch electrode responds to the wall shear stress for smooth wall flow as well as for the disturbed flow region behind the step. When the whole of the surface is electro-active, the response is to mass transfer even when this is not a sole function of wall shear stress. The results demonstrate that the choice of the mass-transfer measurement technique in corrosion studies can have a significant effect on the results obtained from empirical data.

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.

Patch dynamics in grazed subtropical native pastures in south-east Queensland

Patch formation is common in grazed grasslands but the mechanisms involved in the formation and maintenance of patches are not clear. To increase our knowledge on this subject we examined possible reasons for patch formation and the influence of management on changes between patch states in three experiments in native pasture communities in the Crows Nest district, south-east Queensland. In these communities, small-scale patches (tall grassland (dominated by large and medium tussock grasses), short swards (dominated by short tussock grasses and sedges), and lawns (dominated by stoloniferous and/or rhizomatous grasses)) are readily apparent. We hypothesized that the formation of short sward and lawn patches in areas of tall grassland was due to combinations of grazing and soil fertility effects. This was tested in Experiment 1 by applying a factorial combination of defoliation, nutrient application and transplants of short tussock and stoloniferous species to a uniform area of tall grassland. Total species density declined during the experiment, was lower with high nutrient applications, but was not affected by defoliation. There were significant changes in abundance of species that provided support for our hypotheses. With light defoliation and low nutrients, the tall grassland remained dominated by large tussock grasses and contained considerable amounts of forbs. With heavy defoliation, the pastures were dominated by medium tussock grasses and there were significant decreases in forbs and increases in sedges (mainly with low nutrients) and stoloniferous grasses (mainly with high nutrients). Total germinable seed densities and those of most species groups were significantly lower in the heavy defoliation than the light defoliation plots. Total soil seed numbers were not affected by nutrient application but there were fewer seeds of the erect forbs and more sedge seeds in plots with high nutrients. The use of resting from grazing and fire to manage transitions between patches was tested. In Experiment 2, changes in species density and abundance were measured for 5 years in the three patch types with and without grazing. Experiment 3 examined the effects of fire, grazing and resting on short sward patches over 4 years. In Experiment 2, total species density was lower in lawn than short sward or tall grassland patches, and there were more species of erect forbs than other plant groups in all patch types. The lawn patches were originally dominated by Cynodon spp. This dominance continued with grazing but in ungrazed patches the abundance of Cynodon spp. declined and that of forbs increased. In the short sward patches, dominance of short tussock grasses continued with grazing but in ungrazed plots their abundance declined while that of large tussock grasses increased. The tall grassland patches remained dominated by large and medium tussock species. In Experiment 3, fire had no effect on species abundance. On the grazed plots the short tussock grasses remained dominant but where the plots were rested from grazing the small tussock grasses declined and the large tussock grasses increased in abundance. The slow and relatively small changes in these experiments over 4 or 5 years showed how stable the composition of these pastures is, and that rapid changes between patch types are unlikely.

Isolation, solution structure, and insecticidal activity of Kalata B2, a circular protein with a twist: Do Mobius strips exist in nature?

A large number of macrocyclic miniproteins with diverse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using H-1 NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.