COMPLETE ASSIGNMENT OF H-1, C-13 AND N-15 CHEMICAL-SHIFTS FOR BOVINE BETA-LACTOGLOBULIN - SECONDARY STRUCTURE AND TOPOLOGY OF THE NATIVE-STATE IS RETAINED IN A PARTIALLY UNFOLDED FORM

Although beta-lactoglobulin (beta-LG) has been studied extensively for more than 50 years, its physical properties in solution are not yet u nderstood fully in terms of its three-dimensional (3D) structure. For example, despite a recent high-resolution crystal structure, it is sti ll not clear why the two common variants of bovine beta-LG which diffe r by just two residues have different aggregation properties during mi lk processing. We have conducted solution-state NMR studies on a recom binant form of the A variant of beta-LG at low pH conditions where the protein is partially unfolded and exists as a monomer rather than a d imer. Using a C-13, N-15-labelled sample, expressed in Pichia pastoris , we have employed the standard combination of 3D heteronuclear NMR te chniques to obtain near complete assignments of proton, carbon and nit rogen resonances. Using a novel pulse sequence we were able to obtain additional assignments, in particular those of methyl groups in residu es preceding proline within the sequence. From chemical shifts and on the basis of inter-residue NOEs, we have inferred the secondary struct ure and topology of monomeric beta-LG A. It includes eight antiparalle l beta-strands arranged in a barrel, flanked by an alpha-helix, which is typical of a member of the lipocalin family. A detailed comparison with the crystal structure of the dimeric form (for a mixture of A and B variants) at pH 6.5 reveals a close resemblance in both secondary s tructure and overall topology. Both forms have a ninth beta-strand whi ch, at the higher pH, forms part of the dimer interface. These studies represent the first full NMR assignment of beta-LG and will form the basis for a complete characterisation of the solution structure and dy namics of this protein and its variants.