Understanding the role of the L/D-stereospecificity of amino acids is important in obtaining further insight into the mechanism of the formation of amyloid fibrils. Beta(2)-microglobulin is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. A 22-residue peptide of beta(2)-microglobulin, Ser20-Lys41 (L-K3 peptide), obtained by digestion with Acromobacter protease I, formed amyloid-like fibrils in 50% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl at 25 degrees C, as confirmed by thioflavin T fluorescence, circular dichroism spectra, and atomic force microscopy images. A synthetic K3 peptide composed of D-amino acids (D-K3 peptide) formed similar fibrils but with opposite chirality as indicated by circular dichroism spectra. A mixture of L-K3 and D-K3 peptides also formed fibrils, although the L- and D-amino acid composition of each fibril is unknown. To examine the possible cross-reactivity between L- and D-enantiomers, we carried out seeding experiments in which preformed seeds were extended by monomers. The results revealed that only the homologous extensions proceed smoothly, i.e., the growth of L-seeds by L-monomers or D-seeds by D-monomers. The results suggest that, while the fibrils derived from L- and D-peptides form in a similar manner but with opposite stereochemistry, a cross-reaction between them is prevented because the geometry of the mixed sheet cannot satisfy dominant factors for beta-sheet stabilization.

We examined the effects of a fragment of the substrate binding domain of DnaK on protein refolding from chemically denatured states. The fragment DnaK384-638, containing a full-length substrate binding domain, tightly binds to the unfolded protein in solution. The effects of DnaK384-638 on the reactivation of ␤-galactosidase and luciferase were examined at low substrate concentration and low temperature, conditions in which the folding is significantly slow (several days) but the reactivation yield is higher than those in ordinary refolding conditions. In the presence of DnaK384-638, the maximum yield of active ␤-galactosidase was improved from 45% to 65% after a 48-h refolding reaction. Spectroscopic experiments showed that DnaK384-638 bound to partially structured monomers of ␤-galactosidase and consequently suppressed aggregation. DnaK384-638 accelerated the refolding of luciferase to attain equilibrium in 8 h. On the other hand, DnaK386-561, which has no affinity for the substrate, had no chaperone activity for the reactivation of these proteins. These results indicate that the substrate binding of DnaK384-638 facilitates slow protein refolding.T he molecular chaperone DnaK, a member of the HSP70 family in Escherichia coli, is involved in the folding and assembly of newly synthesized polypeptide chains and in preventing the aggregation of stress-denatured proteins. The chaperone activity of DnaK is controlled by ATP (1, 2). When ADP is bound to the N-terminal ATPase domain (residues 1-384), the adjacent C-terminal substrate binding domain (residues 385-638) tightly binds the substrate. ATP bound to the ATPase domain induces a change in the substrate binding domain to a low affinity state for the substrate. The 26-kDa substrate binding domain of DnaK, which contains the ␤-sheet domain and the transitional helix of the substrate binding domain, harbors the substrate binding cleft (residues 393-507) and a 14-kDa domain from residues 508 to 638, of which the first 100 residues are ␣-helical and appear to act as a lid covering the substrate binding cleft (3). Substrate binding occurs by a dynamic mechanism in a two-layered closing device involving independent action of an ␣-helical lid and an arch. Mayer et al. (4) suggested that the ADP-and ATP-bound states of DnaK differ in the frequency of conformational changes that occur in the ␣-helical lid and the ␤-domain that cause an opening of the substrate binding cavity. Previous studies showed that ATP and the cochaperone DnaJ, which stimulate the ATPase activity, are necessary to facilitate the refolding of proteins from chemically denatured states (5, 6).The activity of GroEL, another major chaperone in E. coli, is also controlled by the hydrolysis of ATP and a cochaperone (7,8). However, the monomeric substrate binding domain, minichaperone, alone can assist the refolding of several proteins The chaperone activity of DnaK384-638 was examined for the ability to refold ␤-galactosidase and luciferase from chemically denatured states. These proteins have been used a...

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