Discovery, isolation and structural characterization of cyclotides from Viola sumatrana miq

Cyclotides are cyclic peptides from plants in the Violaceae, Rubiaceae, Fabaceae, Cucurbitaceae, and Solanaceae families. They are sparsely distributed in most of these families, but appear to be ubiquitous in the Violaceae, having been found in every plant so far screened from this family. However, not all geographic regions have been examined and here we report the discovery of cyclotides from a Viola species from South-East Asia. Two novel cyclotides (Visu 1 and Visu 2) and two known cyclotides (kalata S and kalata B1) were identified in V. sumatrana. NMR studies revealed that kalata S and kalata B1 had similar secondary structures. Their biological activities were determined in cytotoxicity assays; both had similar cytotoxic activity and were more toxic to U87 cells compared with other cell lines. Overall, the study strongly supports the ubiquity of cyclotides in the Violaceae and adds to our understanding of their distribution and cytotoxic activity

Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II

MCoTl-I and MCoTl-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTl-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue \#10 within the open-chain variant of MCoTl-II by the non-natural isosteric nucleo amino acid AlaG{[}beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTl-I and MCoTl-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity

Electron capture on ^{8}B nuclei and Superkamiokande results

The energy spectrum of recoil electrons from solar neutrino scattering, as observed by Superkamiokande, is deformed with respect to that expected from SSM calculations. We considered \nu-e scattering from neutrinos produced by the electron-capture on ^{8}B nuclei, e^{-}+^{8}B --> ^{8}Be^{*}+\nu_{e}, as a possible explanation of the spectral deformation. A flux \Phi_{eB}\simeq 10^{4} cm^{-2} s^{-1} could account for Superkamiokande solar neutrino data. However this explanation is untenable, since the theoretical prediction, \Phi_{eB}=(1.3+-0.2) cm^{-2} s^{-1}, is smaller by four orders of magnitude.Comment: 9 pages, incl. 3 figures (epsfig

X-Ray Polarimetry: Historical Remarks and Other Considerations

We briefly discuss the history of X-ray polarimetry for astronomical applications including a guide to the appropriate statistics. We also provide an introduction to some of the new techniques discussed in more detail elsewhere in these proceedings. We conclude our discussion with our concerns over adequate ground calibration, especially with respect to unpolarized beams, and at the system level.Comment: 8 pages, 1 figure presented at "The Coming of Age if X-ray Polarimetry", Rome, Italy April 27, 200

Dissecting the oxidative folding of circular cystine knot miniproteins

Cyclotides are plant proteins with exceptional stability owing to the presence of a cyclic backbone and three disulfide bonds arranged in a cystine knot motif. Accordingly, they have been proposed as templates to stabilize bioactive epitopes in drug-design applications. The two main subfamilies, referred to as the Möbius and bracelet cyclotides, require dramatically different in vitro folding conditions to achieve the native fold. To determine the underlying elements that influence cyclotide folding, we examined the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1. The folding pathways of the two cyclotide subfamilies were found to be different and influenced by specific residues within intercysteine loops 2 and 6. Two changes in these loops, a substitution in loop 2 and an addition in loop 6, enabled the folding of a cycloviolacin O1 analogue under conditions in which folding does not occur in vitro for the native peptide. A key intermediate contains a native-like hairpin structure that appears to be a nucleation locus early in the folding process. Overall, these mechanistic findings on the folding of cyclotides are potentially valuable for the design of new drug leads. Copyright Mary Ann Liebert, Inc

A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted.We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mM, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe(6), and the second involving a charge-charge interaction with the Asp(25) residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules