Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1794, Issue 6, Pages 913-920Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2009.01.009
Keywords
Inhibitor; Enzyme activity; Amyloid fibril; Atomic force microscopy; Thioflavin T; Protein stabilization; SDS PAGE
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Funding
- Council of Scientific and Industrial Research (CSIR), New Delhi
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Inhibiting protein misfolding and aggregation is imperative for treatment of amyloid diseases. in this regard small molecules which bind to and stabilize the monomeric protein have invited attention owing to their ability to significantly slow down or inhibit aggregation and amyloid formation. We have earlier shown that hen egg-white lysozyme (HEWL) spontaneously forms soluble oligomers at pH 12.2, which are later stabilized by intermolecular disulphide bonds, eventually resulting in amyloid fibrils. In this work. we show that overnight (similar to 12 h) pre-incubation of HEWL with its competitive inhibitor, N,N',N ''-Triacetylchitotriose (chitotriose) at neutral pH, impairs its aggregation and fibrillogenesis at pH 12.2. Unlike in control or N-Acetyl-D-glucosamine (NAG) pre-incubated samples, HEWL-chitotriose complex displayed i) reduced thioflavin T and ANS fluorescence, ii) small oligomers but no amyloid fibrils in AFM, iii) absence of large aggregates in SDS-PAGE and gel-filtration elutions, iv) marginally more helical content in CD spectra and v) >70% enzymatic activity after 24 h and similar to 16% activity after week long incubation at alkaline pH. It is likely that strong binding in the HEWL-chitotriose complex, in contrast to weakly bound HEWL-NAG complex, raises the activation energy barrier for protein misfolding and subsequent aggregation, thereby retarding the aggregation kinetics substantially. These results hold promise for the therapy of human lysozyme amyloidosis. (C) 2009 Elsevier B.V. All rights reserved.
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