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Aggregation and fibrillation of beta(2)-microglobulin are hallmarks of dialysis-related amyloidosis. We characterize perturbations of the native conformation of beta(2)-microglobulin that may precede fibril formation. For a beta(2)-microglobulin variant cleaved at lysine 58, we show using capillary electrophoresis that two conformers spontaneously exist in aqueous buffers at neutral pH. Upon treatment of wild-type beta(2)-microglobulin with acetonitrile or trifluoroethanol, two conformations were also observed. These conformations were in equilibrium dependent on the sample temperature and the percentage of organic solvent present. Circular dichroism showed a loss of beta-structures and gain of alpha-helices. Reversal to the native conformation occurred when removing the organics. Affinity capillary electrophoresis experiments showed increased specific interactions of the nonnative beta(2)-microglobulin conformation with the dyes 8-anilino-1-naphthalene sulfonic acid and Congo red. The observations may relate to early folding events prior to amyloid fibrillation and facilitate the development of methods to detect and inhibit pro-amyloid protein and peptide conformations.

Mammalian high mobility group (HMG)-I(Y) chromosomal proteins bind with high affinity to the minor groove of A. T-rich sequences of DNA both in vitro and in vivo. Electrophoretic mobility shift assays demonstrate that in vitro both native and recombinant human HMG-I proteins also bind, but with lower affinity, to preferred regions on isolated avian nucleosome core particles containing approximately 146 base pairs of random sequence DNA. Up to four discrete HMG-I core particle complexes can be detected by electrophoretic mobility shift assays when increasing molar ratios of protein are associated with cores. Both protein-DNA and protein-protein interactions are involved in HMG-I binding to cores. The interaction of HMG-I with core DNA is demonstrated by both thermal denaturation and DNase I footprinting experiments. Chemical cross-linking studies employing reversible photoactivatable cross-linkers, combined with one- and two-dimensional electrophoretic analyses, indicate that in vitro HMG-I binds to cores in close proximity to histones H2A and H2B and H3. In situ cross-linking of K562 human erythroleukemia cell nuclei demonstrate that native HMG-I(Y) binds in a similar manner to nucleosomal histones in vivo. Proteolytic removal of the positively charged amino-terminal tails of the octamer histones abolishes binding of HMG-I to core particles. However, core binding is not mediated by the negatively charged carboxyl-terminal tail of the HMG-I protein since an in vitro produced mutant protein lacking this region binds to core particles in a manner similar to full-length HMG-I. Together these results demonstrate that HMG-I, both in vitro and in vivo, binds to preferred regions on the front face of core nucleosomes.

Chromatin high mobility group protein I (HMG-I) is a mammalian nonhistone protein that has been demonstrated both in vitro and in vivo to preferentially bind to A.T-rich sequences of DNA. Recently the DNA-binding domain peptide that specifically mediates the in vitro interaction of high mobility group protein (HMG)-I with the narrow minor groove of A.T-DNA has been experimentally determined. Because of its predicted secondary structure, the binding domain peptide has been called "the A.T hook" motif. Previously we demonstrated that the A.T hook of murine HMG-I protein is specifically phosphorylated by purified mammalian cdc2 kinase in vitro and that the same site(s) are also phosphorylated in vivo in metaphase-arrested cells. We also found that the DNA binding affinity of short synthetic binding domain peptides phosphorylated in vitro by cdc2 kinase was significantly reduced compared with unphosphorylated peptides. Here we extend these findings to intact natural and recombinant HMG-I proteins. We report that the affinity of binding of full-length HMG-I proteins to A.T-rich sequences is highly dependent on ionic conditions and that phosphorylation of intact proteins by cdc2 kinase reduces their affinity of in vitro binding to A.T-DNA by about 20-fold when assayed near normal mammalian physiological salt concentrations. Furthermore, in cell synchronization studies, we demonstrated that murine HMG-I proteins are phosphorylated in vivo in a cell cycle-dependent manner on the same amino acid residues modified by purified cdc2 kinase in vitro. Together these results strongly support the assertion that HMG-I proteins are natural substrates for mammalian cdc2 kinase in vivo and that their cell cycle-dependent phosphorylation by this enzyme(s) significantly modulates their DNA binding affinity, thereby possibly altering their biological function(s).