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High-throughput identification of novel intrinsically disordered proteins in Arabidopsis thaliana during abiotic stress

Intrinsically disordered proteins and protein regions (IDP/IDR) are a recently recognized group of proteins which are biologically active despite their inherent lack of a well-defined three-dimensional structure. Unlike globular proteins, IDPs lack a unique and stable 3D structure in their native state and exist as fluctuating ensembles of conformations which are well-suited for cellular signaling, stabilization and other adapter functions. In eukaryotes, IDPs have important roles in abiotic stress response. Abiotic stress negatively affects plant growth and yield, and it is of global interest to unravel molecular mechanisms of plant abiotic stress responses. Considering that research on plant IDPs is still at an early stage, the goal of this study was to identify novel IDPs in the model plant Arabidopsis thaliana during cold stress using a combination of in silico and experimental approaches. Cellular extracts were enriched for IDPs by heat denaturation at 99°C which eliminated the majority of globular proteins. IDPs were further separated from remaining thermostable globular proteins using a native/8M urea 2D polyacrylamide gel electrophoresis. Mass spectrometry identified protein sequences from 40 excised gel spots, which were shown to contain 29 different proteins. In order to identify IDPs, sequence analysis of the proteins was conducted using computational disorder predictors PrDOS, DISOPRED3 and MFDp2, which are among the best-performing tools in the field. A total of 12 proteins were classified as IDPs using a consensus-based prediction approach. Gene Ontology analyses demonstrated that the majority of identified proteins had roles in abiotic stress exerted through binding of different partner molecules. Considering the small number of experimentally characterized IDPs at present, the discovery of 12 new IDPs will greatly contribute to current knowledge on intrinsic disorder in plants. As the first high-throughput study of IDPs in a plant proteome of this kind, our research will have a significant impact on the whole IDP field.

adapter molecules, chaperones, disorder predictors, disorder prediction software, cold stress

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