Probing subtle differences in the hydrogen exchange behavior of variants of the human alpha-lactalbumin molten globule using mass spectrometry

The hydrogen-exchange behavior of the low-pH molten globule of human a-lact albumin, containing all four disulfides, has been examined and compared wit h that of a single disulfide variant, [28-111] alpha -lactalbumin, and of a series of proline variants of [28-111] or alpha -actalburnin. The small di fferences in hydrogen-exchange protection exhibited by these partially fold ed species were compared by mixing two or more proteins and monitoring thei r exchange simultaneously using mass spectrometry. helix on hydrogen-exchan ge protection has been investigated using six proline variants of [28-111] alpha -lactalbumin, L11P, L12P, M30P, I95P, K108P and Q117P. The results sh ow that proline mutations in the A, B, C and D alpha -helices lead to a los s of hydrogen-exchange protection for residues in the local helix without p erturbing hydrogen-exchange protection in other regions of the protein. Thu s, local unfolding of the A, B, C and D helices does not significantly alte r the packing and solvent accessibility of other regions of the molten glob ule. By contrast, introduction of a proline residue in the C-terminal 3(10) helix produces a larger and more widespread loss of hydrogen-exchange prot ection, demonstrating that longer-range perturbations of the molten globule have occurred. Thus, residues in this C-terminal region must be involved i n contacts that are critical for the stabilisation of the compact molten gl obule structure. (C) 2001 Academic Press.