Describing 14-3-3 dimerization: tools to obtain KD
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| Year of publication | 2022 |
| Type | Conference abstract |
| Citation | |
| Description | The self-association of proteins is one of the cornerstones of cellular processes. The oligomerization is a fundamental way of protein regulation, influencing their functionality and interactome [1]. Therefore, a strong need for quantitative analysis of oligomerization extent arises when studying proteins. A parameter especially useful for description of self-association is dissociation constant KD [2]. Such constant provides us picture of equilibrium between monomers and higher oligomers, at any given concentration. During our research, we focus closely on 14-3-3 proteins. 14-3-3s are regulators ubiquitous in eukaryotic cells, connected to neurodegenerative and oncologic diseases [3]. In their native state, dimers are formed, crucial for proper function [4]. However, it was revealed that phosphorylation at Ser58 of 14-3-3? leads to monomerization [5,6]. Here, we present an array of biophysical techniques used for oligomeric state quantification of several 14-3-3? constructs: wild type, phosphorylated at Ser58, monomeric and phosphomimicking mutants [5,7]. Contrary to majority of 14-3-3 studies, dimerization description via KD is not biased by used protein concentration. The importance of knowledge of KD is demonstrated, as differences in order of magnitude were discovered [5]. Moreover, workflow for KD determination via 19F Trp NMR is presented, exploiting that tryptophan is not only one of the rarest amino acids, but also has a unique role in protein self-association [8]. |
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