Abstract
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.
Highlights
Accumulation of ordered protein aggregates like amyloid is a symptom of several diseases like Alzheimer’s, Parkinson’s and human lysozyme amyloidosis [1]
Cysteine Carboxymethylation Reaction by Iodoacetamide Iodoacetamide was dissolved in DMF to make a stock solution of 1.5 M. 8 mL of this stock solution of iodoacetamide was directly added in four installments of 2 mL each to 1 mL of hen eggwhite lysozyme (HEWL) sample periodically after completion of 2, 6, 12 and 24 hours of incubation in pH 12.2
Exposed Hydrophobic Regions Probed by ammonium salt (ANS) Previous work has shown that HEWL on exposure to pH 12.2 becomes partially unfolded initiating the process of aggregation [9,13]
Summary
Accumulation of ordered protein aggregates like amyloid is a symptom of several diseases like Alzheimer’s, Parkinson’s and human lysozyme amyloidosis [1]. The role of aberrant disulfide bonds in promoting protein aggregation has assumed significance lately owing to observation of intermolecular disulfide bonds among aggregates of mutant Cu Zn superoxide dismutase (SOD1) in the spinal cord of transgenic mice [2] and in paired helical filaments of tau protein existing as neurofibrillary tangles in human brain [3]. The former is implicated in amyotrophic lateral sclerosis while the latter is the hallmark of Alzheimer’s disease. Several instances where formation of disulfide bonds can alter the aggregation pathway [5], reduce toxicity of amyloid fibrils [6] and change material properties of the amyloid fibrils [5] have been reported
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