STRUCTURAL CHARACTERIZATION AND COMPARISON OF THE NATIVE AND A-STATESOF EQUINE LYSOZYME

Native state H-1 NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrog en atoms in the native state. Native hole equine lysozyme hydrogen exc hange protection factors are as large as 10(6), the most protected res idues being located in elements of secondary structure. High exchange protection in the domain interface correlates with the binding of Ca2 in this region. Equine lysozyme differs from most non-Ca2+ binding ly sozymes in forming a highly populated partially folded state at low PH . The protein in this A-state at PH 2.0 has been found to bind 1-anili no-naphthalene-8-sulphonate with the enhancement of fluorescent intens ity and blue shift in the spectral maximum characteristic of molten gl obules. NMR spectra indicate that the A-state is globally much less or dered than native equine lysozyme but does not contain significant reg ions of random coil structure. The amides most protected against hydro gen exchange in the A-state (protection factors up to 10(2) at 5 degre es C) correspond to residues of three of the four alpha-helices of the native state; the side-chains of these residues form a hydrophobic cl uster that includes five aromatic residues. Circular dichroism and try ptophan fluorescence indicate that these residues are substantially mo re constrained than similar residues in ''classical'' molten globules. Taken together, the data suggest a model for the A-state of equine ly sozyme in which a more ordered core is surrounded by a less ordered bu t still compact polypeptide chain. (C) 1997 Academic Press Limited.