NDLI: On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

Content Provider	PubMed Central
Author	Ravi, Vijay Kumar Swain, Tulsi Chandra, Nividh Swaminathan, Rajaram
Editor	Khan, Rizwan H.
Copyright Year	2014
Abstract	Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6±0.6)×10−5 s−1). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.
Related Links	http://dx.doi.org/10.1371/journal.pone.0087256
Starting Page	87256
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	1
Volume Number	9
Language	English
Publisher	Public Library of Science
Publisher Date	2014-01-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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