1c2u
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(New page: 200px<br /><applet load="1c2u" size="450" color="white" frame="true" align="right" spinBox="true" caption="1c2u" /> '''SOLUTION STRUCTURE OF [ABU3,35]SHK12-28,17-3...)
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Revision as of 10:00, 20 November 2007
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SOLUTION STRUCTURE OF [ABU3,35]SHK12-28,17-32
Overview
ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla, helianthus, is a 35 residue polypeptide cross-linked by three disulfide, bridges: Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. To investigate the role, of these disulfides in the structure and channel-blocking activity of ShK, toxin, a series of analogues was synthesized by selective replacement of, each pair of half-cystines with two alpha-amino-butyrate (Abu) residues., The remaining two disulfide pairs were formed unambiguously using an, orthogonal protecting group strategy of Cys(Trt) or Cys(Acm) at the, appropriate position. The peptides were tested in vitro for their ability, to block Kv1.1 and Kv1.3 potassium channels and their ability to displace, [(125)I]dendrotoxin binding to rat brain synaptosomal membranes. The, monocyclic peptides showed no activity in these assays. Of the dicyclic, peptides, [Abu12,28]ShK(3-35,17)(-)(32) (where the subscript indicates, disulfide connectivities) had weak activity on Kv1.3 and Kv1.1., [Abu17,32]ShK(3-35,12)(-)(28) blocked Kv1.3 with low nanomolar potency, but was less effective (being comparable to [Abu12,28]ShK(3-35,17)(-)(32)), against Kv1.1. [Abu3, 35]ShK(12-28,17)(-)(32), retained high picomolar, affinity against both channels. Corroborating these results, [Abu3,35]ShK(12-28, 17)(-)(32) had an IC(50) ratio relative to native, toxin of 18 in the displacement assay, whereas, [Abu17,32]ShK(3-35,12)(-)(28) and [Abu12, 28]ShK(3-35,17)(-)(32) had, ratios of 69 and 390, respectively. Thus, the disulfide bond linking the, N- and C-terminal regions is less important for activity than the internal, disulfides. NMR analysis of the [Abu12,28] and [Abu17,32] analogues, indicated that they had little residual structure, consistent with their, significantly reduced activities. By contrast, [Abu3,35]ShK(12-28,17)(-)(32) had a moderately well-defined solution, structure, with a mean pairwise root-mean-square deviation of 1.33 A over, the backbone heavy atoms. This structure nevertheless showed significant, differences from that of native ShK toxin. The possible interactions of, this analogue with the channel and the distinction between native, secondary and tertiary structure on one hand and global topology imposed, by the disulfide bridges on the other are discussed.
About this Structure
1C2U is a Single protein structure of sequence from [1]. Full crystallographic information is available from OCA.
Reference
Role of disulfide bonds in the structure and potassium channel blocking activity of ShK toxin., Pennington MW, Lanigan MD, Kalman K, Mahnir VM, Rauer H, McVaugh CT, Behm D, Donaldson D, Chandy KG, Kem WR, Norton RS, Biochemistry. 1999 Nov 2;38(44):14549-58. PMID:10545177
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