Suppression of lysozyme aggregation at alkaline pH by tri-N-acetylchitotriose

Kumar, Satish; Ravi, Vijay; Swaminathan, Rajaram

doi:10.1016/j.bbapap.2009.01.009

Cited by 35 publications

(28 citation statements)

References 44 publications

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“…Amyloid fibrils with extensive branching, a rare event, were also observed as early as 12 hours after incubation at pH 12.2 (image D). Branching among HEWL amyloid fibrils has been observed by us previously too [28]. Interestingly, image C shows tiny straight and pointed fibril-like structures emanating from all amorphous aggregates.…”

Section: Resultssupporting

confidence: 70%

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

et al. 2014

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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.

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Section: Resultssupporting

confidence: 70%

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

et al. 2014

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“…Concerning the multi-step process of protein aggregation, several therapeutic approaches have been invented to prevent or to treat the amyloid-affected individuals [14,23]. These approaches mainly include direct inhibition of the self-assembly processes, attenuating the production of amyloidogenic forms, attenuating the stability of the native structure of proteins and finally raising the clearance rate of aggregated proteins [24].…”

Section: Introductionmentioning

confidence: 99%

Anti-amyloidogenic and fibril-destabilizing effects of two manganese–salen derivatives against hen egg-white lysozyme aggregation

Bahramikia

Yazdanparast

2012

International Journal of Biological Macromolecules

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“…Previous work has shown that HEWL on exposure to pH 12.2 becomes partially unfolded initiating the process of aggregation [9], [13]. Exposed hydrophobic regions among the aggregates were monitored using ANS, which serves as an indicator for the growth of aggregates and its suppression [14].…”

Section: Resultsmentioning

confidence: 99%

Preventing Disulfide Bond Formation Weakens Non-Covalent Forces among Lysozyme Aggregates

et al. 2014

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Nonnative disulfide bonds have been observed among protein aggregates in several diseases like amyotrophic lateral sclerosis, cataract and so on. The molecular mechanism by which formation of such bonds promotes protein aggregation is poorly understood. Here in this work we employ previously well characterized aggregation of hen eggwhite lysozyme (HEWL) at alkaline pH to dissect the molecular role of nonnative disulfide bonds on growth of HEWL aggregates. We employed time-resolved fluorescence anisotropy, atomic force microscopy and single-molecule force spectroscopy to quantify the size, morphology and non-covalent interaction forces among the aggregates, respectively. These measurements were performed under conditions when disulfide bond formation was allowed (control) and alternatively when it was prevented by alkylation of free thiols using iodoacetamide. Blocking disulfide bond formation affected growth but not growth kinetics of aggregates which were ∼50% reduced in volume, flatter in vertical dimension and non-fibrillar in comparison to control. Interestingly, single-molecule force spectroscopy data revealed that preventing disulfide bond formation weakened the non-covalent interaction forces among monomers in the aggregate by at least ten fold, thereby stalling their growth and yielding smaller aggregates in comparison to control. We conclude that while constrained protein chain dynamics in correctly disulfide bonded amyloidogenic proteins may protect them from venturing into partial folded conformations that can trigger entry into aggregation pathways, aberrant disulfide bonds in non-amyloidogenic proteins (like HEWL) on the other hand, may strengthen non-covalent intermolecular forces among monomers and promote their aggregation.

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Suppression of lysozyme aggregation at alkaline pH by tri-N-acetylchitotriose

Cited by 35 publications

References 44 publications

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

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

Anti-amyloidogenic and fibril-destabilizing effects of two manganese–salen derivatives against hen egg-white lysozyme aggregation

Preventing Disulfide Bond Formation Weakens Non-Covalent Forces among Lysozyme Aggregates

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