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J.F. Koretz, C. Eifert, M. Burgio, P. Bennett, J. Salerno; N–Terminal Control of Small Heat Shock Protein Aggregate Size and Chaperone–Like Activity Illustrated with AlphaA–Crystallin and Mj Hsp16.5 . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3379.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To characterize further the relationship between small heat shock protein N–terminal length and sequence with aggregate oligomerization and functionality, building upon our initial study of N–terminal swapping between alphaA–crystallin and Mj Hsp16.5 (Salerno et al., Prot. Eng., 2003). Methods: Chimera designs included the addition of the following N–terminal sequences to the central core and C–terminal region of alphaA–crystallin – the N–terminal of Mj Hsp16.5; the N–terminal sequence of Mj Hsp16.5 reversed in order; and a selected sequence from nNOS of approximately the same length and hydropathicity as the N–terminal of alphaA–crystallin. The N–terminal of alphaA–crystallin was added to the central core and C–terminal of Mj Hsp16.5. These four chimeras were compared to the native proteins and to sequences without an added N–terminal for aggregate size, shape, and functionality, using FPLC, EM, and the insulin dissociation assay respectively. Results: Chimera aggregate size and shape were functions of both N–terminal length and N–terminal sequence, while chaperone–like activity was preserved for all species with the core and C–terminal of alphaA–crystallin. The core and C–terminal of Mj Hsp16.5 are not well expressed in the absence of an N–terminal, and are not active when the alphaA N–terminal sequence is substituted for the native N–terminal. Conclusions: N–terminal sequence length and properties play major roles in determining final aggregate size. For heat shock proteins such as alpha–crystallin, which are polydisperse, the N–terminal is a much less critical factor in aggregation and function than in the monodisperse particles which rely on precise alignment of subunits for both assembly and functionality.
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