A non-natural variant of human lysozyme (I59T) mimics the in vitro behaviour of the I56T variant that is responsible for a form of familial amyloidosis

Abstract

We report here the detailed characterisation of a non-naturally occurring variant of human lysozyme, I59T, which possesses a destabilising point mutation at the interface of the alpha- and beta-domains. Although more stable in its native structure than the naturally occurring variants that give rise to a familial form of systemic amyloidosis, I59T possesses many attributes that are similar to these disease-associated species. In particular, under physiologically relevant conditions, I59T populates transiently an intermediate in which a region of the structure unfolds cooperatively; this loss of global cooperativity has been suggested to be a critical feature underlying the amyloidogenic nature of the disease-associated lysozyme variants. In the present study, we have utilised this variant to provide direct evidence for the generic nature of the conformational transition that precedes the ready formation of the fibrils responsible for lysozyme-associated amyloid disease. This non-natural variant can be expressed at higher levels than the natural amyloidogenic variants, enabling, for example, singly isotopically labelled protein to be generated much more easily for detailed structural studies by multidimensional NMR spectroscopy. Moreover, we demonstrate that the I59T variant can readily form fibrils in vitro, similar in nature to those of the amyloidogenic I56T variant, under significantly milder conditions than are needed for the wild-type protein.

The Significance of the Location of Mutations for the Native-State Dynamics of Human Lysozyme

“…The thermodynamic parameters were computed under the assumption of a twostate model for the unfolding reaction. The corrected fluorescence intensity at 355 nm was plotted against the GdnHCl concentration in each sample, and the data were fit as described in (19) …”

“…EX1 HD exchange of variants was monitored by electrospray ionization MS (pH 8, 37 C or 47 C) and the data were processed as previously described (19,20). Proteins were deuterated at exchangeable sites by dissolving lysozyme (0.2 mg) in D 2 O (pH 3.8, 350 mL), followed by heating (70 C, 10 min) and then cooling to room temperature.…”

“…The samples were immediately placed in a (19,38), and the threshold for significant chemical-shift differences was fixed at 0.1 ppm (19,21). Red arrows indicate the sites of mutation.…”

“…The transient intermediate species can also be detected under similar conditions in the nonnatural variant, I59T, which has a native-state stability lying between that of the I56T variant and WT. Interestingly, the transient intermediate species can be detected in both the T70N variant and WT, but only under more destabilizing conditions (47 C and 57 C, respectively) and not under conditions that are physiologically relevant (19,20).…”

“…In this study, we set out to examine how the introduction of alternative mutations would modify the properties of lysozyme, and for this purpose we selected previously reported, well-expressed lysozyme variants (I56V and I89V (26), I23A (27), and I59T (16,19,28)). The I23A variant was chosen because this a-domain mutation affects the native-state stability of the protein to a similar degree as the well-characterized I59T (b-domain) variant (19), allowing us to directly compare the effect of a mutation in the a-domain relative to one in the b-domain. We also chose a mutation that does not significantly destabilize the native state of lysozyme but is located in the critical domain interface region, I56V.…”