Structural elucidation of a hydrophobic box in bovine .alpha.-lactalbumin by NMR: nuclear Overhauser effects

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Abstract
The proton nuclear Overhauser effects of bovine .alpha.-lactalbumin were studied at 2000 MHz by irradiation of an upfield ring current shifted methylene at -2.45 ppm (assigned to Ile-95) and two aromatic protons, Tyr-103 (8.36 ppm) and Trp-60 (5.85 ppm). The experimental results were consistent with a putative three-dimensional .alpha.-lactalbumin model [Warme, P. K., Momany, F. A., Rumball, S. V., Tuttle, R. W., and Scheraga, H. A. (1974) Biochemistry 13, 768-782], which predicted the close proximity of Ile-95, Tyr-103, Trp-60, and Trp-104. Several of the assignments correlated with those previously made from cheimcally induced dynamic nuclear polarization experiments [Berliner, L. J., and Kaptein, R. (1981) Biochemistry 20, 799-807]. Subtle differences in the structure of this hydrophobic box region in .alpha.-lactalbumin were found between the Ca(II) and apo forms of the protein. The existence of this "hydropobic box" in .alpha.-lactalbumin was strikingly similar to that in lysozyme, as verified in solution.