Beta-lactoglobulin (beta-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly beta structure. The structural function of the only alpha-helix with three turns at the C-terminus is unknown. Vitamin D(3) binds to the central calyx formed by the beta-strands. Whether there are two vitamin D binding-sites in each beta-LG molecule has been a subject of controversy. Here, we report a second vitamin D(3) binding site identified by synchrotron X-ray diffraction (at 2.4 A resolution). In the central calyx binding mode, the aliphatic tail of vitamin D(3) clearly inserts into the binding cavity, where the 3-OH group of vitamin D(3) binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 A). The second binding site, however, is near the surface at the C-terminus (residues 136-149) containing part of an alpha-helix and a beta-strand I with 17.91 A in length, while the span of vitamin D(3) is about 12.51 A. A remarkable feature of the second exosite is that it combines an amphipathic alpha-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a beta-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D(3) binding. This finding provides a new insight into the interaction between vitamin D(3) and beta-LG, in which the exosite may provide another route for the transport of vitamin D(3) in vitamin D(3) fortified dairy products. Atomic coordinates for the crystal structure of beta-LG-vitamin D(3) complex described in this work have been deposited in the PDB (access code 2GJ5).

We use scanning tunneling spectroscopy to explore the quantum well states in the Pb islands grown on a Cu(111) surface. Our observation demonstrates that the empty quantum well states, whose energy levels lie beyond 1.2 eV above the Fermi level, are significantly affected by the image potential. As the quantum number increases, the energy separation between adjacent states is shrinking rather than widening, contrary to the prediction for a square potential well. By simply introducing a phase factor to reckon the effect of the image potential, the shrinking behavior of the energy separation can be reasonably explained with the phase accumulation model. The model also reveals that there exists a quantum regime above the Pb surface in which the image potential is vanished. Moreover, the quasi-image-potential state in the tunneling gap is quenched because of the existence of the quantum well states.

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