A polypeptide with 42 amino acids, SA-42, has been designed to fold into a 'hairpin' helix-loophelix motif. Its intended use is to serve as a template for the rational design of catalytically active polypeptides. SA-42 is made up of two amphiphilic helices, that are designed to interact through the hydrophobic effect and are connected by a short loop. Evidence is presented that it does adopt the intended motif and that it dimerises in an antiparallel mode at concentrations between 2 pmol dm-3 and 5.6 mmol dmP3. The structural evidence was obtained from NMR and CD spectroscopy and the state of aggregation was determined with equilibrium sedimentation ultracentrifugation and CD spectroscopy. Special attention was paid to the design of SA-42 with respect to the expected NMR spectrum. The mean residue ellipticity at 222 nm of SA-42, -25 000 deg cm2 dmol-', compares well with that of other published helix-loophelices. Two polypeptides that were developed from SA-42 by the replacement of three and five residues, respectively, were recently shown to have catalytical activity .

Abstract-Direct binding of apolipoprotein (apo)B-containing lipoproteins to proteoglycans is the initiating event inatherosclerosis, but the processes involved at later stages of development are unclear. Here, we investigated the importance of the apoB-proteoglycan interaction in the development of atherosclerosis over time and investigated the role of lipoprotein lipase (LPL) to facilitate low-density lipoprotein (LDL) retention at later stages of development. Atherosclerosis was analyzed in apoB transgenic mice expressing LDL with normal (control LDL) or reduced proteoglycan-binding (RK3359-3369SA LDL) activity after an atherogenic diet for 0 to 40 weeks. The initiation of atherosclerosis was delayed in mice expressing RK3359-3369SA LDL, but they eventually developed the same level of atherosclerosis as mice expressing control LDL. Retention studies in vivo showed that although higher levels of 131 I-tyramine cellobiose-labeled control LDL ( 131 I-TC-LDL) were retained in nonatherosclerotic aortae compared with RK3359-3369SA 131 I-TC-LDL, the retention was significantly higher and there was no difference between the groups in atherosclerotic aortae. Lower levels of control 125 I-TC-LDL and RK3359-3369SA 125 I-TC-LDL were retained in atherosclerotic aortae from ldlr Ϫ/Ϫ mice transplanted with lpl Ϫ/Ϫ compared with lpl ϩ/ϩ bone marrow. Uptake of control LDL or RK3359-3369SA LDL into macrophages with specific expression of human catalytically active or inactive LPL was increased compared with control macrophages. 1 We tested the response-toretention hypothesis 2 in an earlier study using genetically modified mice that expressed human recombinant lowdensity lipoproteins (LDLs) with reduced proteoglycanbinding activity and provided direct evidence showing that subendothelial retention of apolipoprotein (apo)B100-containing lipoproteins is the initiating event in atherogenesis. 3 Furthermore, we demonstrated that the atherogenicity of LDL is linked to their proteoglycan-binding activity. 3 Lipoproteins associate with artery wall proteoglycans via both direct and indirect interactions. Direct binding between LDL and proteoglycans involves an ionic interaction between basic amino acids in apoB100 (Site B; residues 3359 to 3369) and negatively charged sulfate groups on the glycosaminoglycan (GAG) chains of proteoglycans. 4,5 Indirect binding between LDL and proteoglycans is facilitated by apoE, 3 which is found in human atherosclerotic plaques together with apoB. 6 ApoE binds vascular proteoglycans, and apoEenrichment of proteoglycan-binding-defective LDL (in which Site B has been inactivated by mutagenesis) partly restores proteoglycan binding. 3 Mouse LDL contains significant amounts of apoE, and consequently proteoglycan-bindingdefective LDL isolated from mouse plasma displays Ϸ30% of normal proteoglycan binding. 3 Subendothelial retention of LDL via indirect binding to GAGs can also be facilitated by lipoprotein lipase (LPL). 7 We have shown previously that the binding between LPL and LDL is mediated throug...

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Detailed information about molten globule structures in aqueous solution can be obtained from NMR spectroscopy if the spectra are assigned in dilute TFE solution. On the basis of the NMR spectroscopic analysis, the solution structure of SA-42 was found to be close to the designed one. A route for developing native-like properties in SA-42 is suggested based on the identification by NMR spectroscopy of some less well ordered amino acid sidechains in the hydrophobic core and on the observed structural rigidity of the two helices.