Using genetic engineering technologies, the chitin-binding domain (ChBD) of the human macrophage chitotriosidase has been inserted into the host protein BlaP, a class A b-lactamase produced by Bacillus licheniformis. The product of this construction behaved as a soluble chimeric protein that conserves both the capacity to bind chitin and to hydrolyze b-lactam moiety. Here we describe the biochemical and biophysical properties of this protein (BlaPChBD). This work contributes to a better understanding of the reciprocal structural and functional effects of the insertion on the host protein scaffold and the heterologous structured protein fragments. The use of BlaP as a protein carrier represents an efficient approach to the functional study of heterologous protein fragments.Keywords: b-Lactamase; domain insertion; protein engineering; hybrid protein; chitin-binding domain; protein stability For a long time, the structure, stability, and function of a protein have been predominantly associated with its primary structure (Tsybovsky et al. 2004). Modifications of the primary structure by point mutations have been investigated intensively and are known to alter the stability and the activity of the protein to various degrees (Hirabayashi and Kasai 1991;Lee and Levitt 1991;Palzkill and Botstein 1992;Tepper et al. 1994;Rouse et al. 1996). The effects of peptide insertions on the structure and function of proteins have, in contrast, not been investigated in detail yet (Betton et al. 1997;Collinet et al. 2000).Peptide insertions can occur naturally as a consequence of genetic rearrangement resulting in the introduction of an amino acid sequence within an unrelated one. Such a phenomenon has been observed in large natural proteins made of at least two structural domains (Collinet et al. 2000). The result of these genetic rearrangements is the presence of discontinuous domains in natural proteins, where the linear sequence of one domain is interrupted by another inserted one. A systematic survey, indeed, indicates that 28% of structural domains are discontinuous (Jones et al. 1998). A few examples of such phenomena are found in DsbA (Martin et al. 1993), DNA polymerase Reprint requests to: Moreno Galleni, Macromolécules Biologiques, Centre d'Ingénierie des Protéines, Université de Liège, Institut de Chimie B6a, Sart Tilman, Ulg, B-4000 Liège, Belgium; e-mail: mgalleni@ulg.ac.be; fax: 32-4-3663364.Abbreviations: ChBD, chitin-binding domain of the human macrophage chitotriosidase; ESMS, electrospray ionization mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; DTT, dithiothreitol; DTNB, dithiobis-(2-nitrobenzoic acid); BSA, bovine serum albumin; ANS, 1-anilino-8-naphthalenesulfonate.Article and publication are at

Nine neurodegenerative disorders, called polyglutamine (polyQ) diseases, are characterized by the formation of intranuclear amyloid-like aggregates by nine proteins containing a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis. The mechanism by which polyQ expansions trigger the aggregation of the relevant proteins remains, however, unclear. In this work, polyQ tracts of different lengths were inserted into a solvent-exposed loop of the β-lactamase BlaP and the effects of these insertions on the properties of BlaP were investigated by a range of biophysical techniques. The insertion of up to 79 glutamines does not modify the structure of BlaP; it does, however, significantly destabilize the enzyme. The extent of destabilization is largely independent of the polyQ length, allowing us to study independently the effects intrinsic to the polyQ length and those related to the structural integrity of BlaP on the aggregating properties of the chimeras. Only chimeras with 55Q and 79Q readily form amyloid-like fibrils; therefore, similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the chimeras aggregate into amyloid-like fibrils. Most importantly, the chimera containing 79Q forms amyloid-like fibrils at the same rate whether BlaP is folded or not, whereas the 55Q chimera aggregates into amyloid-like fibrils only if BlaP is unfolded. The threshold value for amyloid-like fibril formation depends, therefore, on the structural integrity of the β-lactamase moiety and thus on the steric and/or conformational constraints applied to the polyQ tract. These constraints have, however, no significant effect on the propensity of the 79Q tract to trigger fibril formation. These results suggest that the influence of the protein context on the aggregating properties of polyQ disease-associated proteins could be negligible when the latter contain particularly long polyQ tracts.