Role of α-β transition in the folding of proteins

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The α-helix to β-sheet(α→β)transition of proteins is a key issue to understand the folding and biological function of a number of proteins. Folding of β-lactoglobulin is a useful model for clarifying the mechanism of the α→β transition because the kinetic intermediate contains non-native α-helical structure. With recombinant bovine β-lactoglobulin A uniformly labeled with ^<15>N and heteronuclear NMR spectroscopy, we analyzed its folding kinetics and dynamics.1. The H/D exchange experiments have been performed on the native state, demonstrating the presence of a stable hydrophobic core.2. To define the structural and dynamic properties of an early folding intermediate in β-lactoglobulin, the kinetics of folding was measured, using ultra-rapid mixing techniques in conjunction with hydrogen exchange labeling probed by heteronuclear NMR.We demonstrated that, in the early kinetic intermediate, the non-native α-helix is formed at the N-terminal region of the molecule.3. We studied the conformation and stability of the various forms of β-lactoglobulin by heteronuclar NMR.We showed that the modification of the buried thiol group of β2-microglobulin destabilizes the entire parts of the molecule.4. To understand the mechanism of amyloid formation, which is proposed to include the α→β transition, we expressed human β2-microglobulin in the methylotropic yeast, Pichia pastoris. The recombinant β2-microglobulin formed amyloid fibrils as demonstrated by electron microscopy and atomic force microscopy. With fluorescence microscopy, we observed the amyloid fibrils made of β2-microglobulin and its extension process.