Dataset Information

Effect of N-Ethylmaleimide as a Blocker of Disulfide Crosslinks Formation on the Alkali-Cold Gelation of Whey Proteins.

ABSTRACT: N-ethylmaleimide (NEM) was used to verify that no new disulfide crosslinks were formed during the fascinating rheology of the alkali cold-gelation of whey proteins, which show Sol-Gel-Sol transitions with time at pH > 11.5. These dynamic transitions involve the formation and subsequent destruction of non-covalent interactions between soluble whey aggregates. Therefore, incubation of aggregates with NEM was expected not to affect much the rheology. Experiments show that very little additions of NEM, such as 0.5 mol per mol of protein, delayed and significantly strengthened the metastable gels formed. Interactions between whey protein aggregates were surprisingly enhanced during incubation with NEM as inferred from oscillatory rheometry at different protein concentrations, dynamic swelling, Trp fluorescence and SDS-PAGE measurements.

SUBMITTER: Lei Z

PROVIDER: S-EPMC5061392 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Effect of N-Ethylmaleimide as a Blocker of Disulfide Crosslinks Formation on the Alkali-Cold Gelation of Whey Proteins.

Lei Zhao Z Chen Xiao Dong XD Mercadé-Prieto Ruben R

PloS one 20161012 10

N-ethylmaleimide (NEM) was used to verify that no new disulfide crosslinks were formed during the fascinating rheology of the alkali cold-gelation of whey proteins, which show Sol-Gel-Sol transitions with time at pH > 11.5. These dynamic transitions involve the formation and subsequent destruction of non-covalent interactions between soluble whey aggregates. Therefore, incubation of aggregates with NEM was expected not to affect much the rheology. Experiments show that very little additions of N ...[more]

PMID: 27732644

Similar Datasets

Project description:The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.

Dataset Information

Effect of N-Ethylmaleimide as a Blocker of Disulfide Crosslinks Formation on the Alkali-Cold Gelation of Whey Proteins.

Publications

Effect of N-Ethylmaleimide as a Blocker of Disulfide Crosslinks Formation on the Alkali-Cold Gelation of Whey Proteins.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets