Mechanistic Studies of the Folding of Human Lysozyme and the Origin of Amyloidogenic Behavior in Its Disease-Related Variants

Abstract

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.

The Extracellular Chaperone Clusterin Potently Inhibits Human Lysozyme Amyloid Formation by Interacting with Prefibrillar Species

“…In-depth characterization of two of the disease-associated variants, I56T and D67H, as well as the non-diseaserelated T70N variant, has been performed using a variety of biophysical techniques. [7][8][9][10][11][12][13][14] Deposits of I56T and D67H formed in vitro possess the key characteristics of amyloid fibrils; these include their fibrillar morphology in TEM images, their displays of birefringence upon binding the dye Congo red and a typical cross-β X-ray diffraction pattern. [10][11][12] The kinetics of in vitro fibril formation by both I56T and D67H are sigmoidal, showing a lag phase followed by an exponential growth phase typical of amyloidogenic systems.…”

Structural and Folding Dynamic Properties of the T70N Variant of Human Lysozyme

“…Besides being, in fact, one of the best characterized enzymes, its fibrillogenic mechanism is not influenced by protein fragmentation; nor, to our knowledge, does the wild-type species generate amyloid deposits in vivo, even in the elderly. Thorough analysis of several biochemical properties of the amyloidogenic variants in comparison to the wildtype species showed that pathogenic lysozymes are less stable than wild-type (3)(4)(5)(6)(7)(8). This thermodynamic destabilization correlates with an increased concentration of partly unfolded intermediates that self-aggregate into fibrillar polymers.…”

Monitoring disappearance of monomers and generation of resistance to proteolysis during the formation of the activation domain of human procarboxypeptidase A2 (ADA2h) amyloid fibrils by matrix-assisted laser-desorption ionization–time-of-flight-MS

“…The main resultant peptide in the mass spectrum spans from the N-terminus of the protein to an internal part of α-helix 1, the end product of CPY digestion. This indicates that before fibril formation the protein unfolds partially, as has been postulated for other proteins with amyloid propensity [16][17][18]. In fact, we have demonstrated previously that when we changed the pH by dialysis from 7.0 to 3.0 in a diluted sample that was not submitted to high temperature, as is the case in this experiment, the CD spectrum corresponded to an αβ-conformation with an increase in the random-coil conformation (the minimum at 208 nm is increased) [6].…”