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Figure 2
Cartoon representation of the construct-design process. (a) For multi-domain proteins, the domain of interest is identified using experimental and/or alignment data. (b) The domain architecture of the isolated domain is then inspected for suitable start and end points (bright green), avoiding cutting through secondary-structure elements (magenta) and with the aim of including all residues required for function. Limited proteolysis data as well as secondary-structure prediction tools can be used as a guide, as well as structural data of homologue proteins. Sample homogeneity can be achieved at the sequence level through the mutation of residues targeted by post-translational modifications (PTMs) such as phosphorylation (orange) that either prevent or mimic the PTM. Further construct optimization can involve the mutation of surface residues with flexible or charged side chains (cyan), either as single mutants or in clusters, to alanine or residue types that reverse or remove the charge. Regions of low structural complexity (grey, dark green) can be replaced by short linker residues or equivalent residues in homologue proteins to reduce conformational variability in the construct. (c) Events such as cofactor binding or PTMs can affect the conformational state and ligand-binding ability of the protein and should be considered in the design of the experiment.

Journal logoSTRUCTURAL
ISSN: 2059-7983
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