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 alpha-lactalbumin, containing all four disulfides, has been examined and compared with that of a single disulfide variant, [28-111] alpha-lactalbumin, and of a series of proline variants of [28-111] alpha-lactalbumin. The small differences in hydrogen-exchange protection exhibited by these partially folded species were compared by mixing two or more proteins and monitoring their exchange simultaneously using mass spectrometry. The effect of single proline mutations within each alpha-domain helix on hydrogen-exchange protection has been investigated using six proline variants of [28-111] alpha-lactalbumin, L11P, L12P, M30P, I95P, K108P and Q117P. The results show that proline mutations in the A, B, C and D alpha-helices lead to a loss of hydrogen-exchange protection for residues in the local helix without perturbing hydrogen-exchange protection in other regions of the protein. Thus, local unfolding of the A, B, C and D helices does not significantly alter the packing and solvent accessibility of other regions of the molten globule. By contrast, introduction of a proline residue in the C-terminal 3(10) helix produces a larger and more widespread loss of hydrogen-exchange protection, demonstrating that longer-range perturbations of the molten globule have occurred. Thus, residues in this C-terminal region must be involved in contacts that are critical for the stabilisation of the compact molten globule structure.

Protein folding and interactions revealed by mass spectrometry.

Mass spectrometry is capable of examining very large, dynamic proteins and this ability, coupled with its relatively high throughput and low sample requirements, is reflected by its increasing importance for the characterisation of protein structure. Recent developments in mass spectrometry, in particular the refinement of the electrospray process and its coupling with time-of-flight mass analysis, mean that it is poised to contribute not only as a complementary tool but also with a defined role in many areas of chemical biology.

Mechanistic studies of the folding of human lysozyme and the origin of amyloidogenic behavior in its disease-related variants.

The unfolding and refolding properties of human lysozyme and two amyloidogenic variants (Ile56Thr and Asp67His) have been studied by stopped-flow fluorescence and hydrogen exchange pulse labeling coupled with mass spectrometry. The unfolding of each protein in 5.4 M guanidine hydrochloride (GuHCl) is well described as a two-state process, but the rates of unfolding of the Ile56Thr variant and the Asp67His variant in 5.4 M GuHCl are ca. 30 and 160 times greater, respectively, than that of the wild type. The refolding of all three proteins in 0.54 M GuHCl at pH 5.0 proceeds through persistent intermediates, revealed by multistep kinetics in fluorescence experiments and by the detection of well-defined populations in quenched-flow hydrogen exchange experiments. These findings are consistent with a predominant mechanism for refolding of human lysozyme in which one of the structural domains (the alpha-domain) is formed in two distinct steps and is followed by the folding of the other domain (the beta-domain) prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67His variant are closely similar to those of the wild-type protein, consistent with the location of this mutation in an outer loop of the beta-domain which gains native structure only toward the end of the refolding process. By contrast, the Ile56Thr mutation is located at the base of the beta-domain and is involved in the domain interface. The refolding of the alpha-domain is unaffected by this substitution, but the latter has the effect of dramatically slowing the folding of the beta-domain and the final assembly of the native structure. These studies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in the stability of the native fold relative to partially folded intermediates. The origin of this instability is different in the two variants, being caused in one case primarily by a reduction in the folding rate and in the other by an increase in the unfolding rate. In both cases this results in a low population of soluble partially folded species that can aggregate in a slow and controlled manner to form amyloid fibrils.