Predictable tuning of hydrogel systems is achieved by detailed analysis of higher-order network formation of well-defined multidomain components ( Figure 1E). Fundamental understanding of the composition and formation of liquid–liquid phase separation (LLPS) of signaling molecules into microscale biomolecular condensates can be achieved by analysis of synthetic LLPS systems ( Figure 1D). Synthetic multienzyme assemblies are applied to optimize enzyme reaction rates and reaction efficiency ( Figure 1C). In synthetic signaling pathways, in vivo introduction of single synthetic modules allows for analysis of plasticity of native signaling pathways and the introduction of new functionalities ( Figure 1B). (24) Figure 1Įngineered MDSPs have been applied across multiple length scales in various fields of interest for different purposes. (20,21) Fundamental insight into these mechanisms reveals how conjugated protein domains, forming engineered scaffold proteins, can be used as regulators of in- and output of signaling pathways (22,23) and generates understanding of their higher-order assembly into networks. These precisely designed synthetic platforms aid elucidation of various molecular mechanisms, such as plasticity of pathways, (13,14) evolutionary recombination, (15,16) nonlinearity in signaling output, (17,18) phase transition, (19) and the effect of multivalency. Libraries consisting of well-defined synthetic modules have been generated by conjugation, either through multimerization or recombination of native scaffold domains ( Figure 1A). Here, we highlight this second class of conjugated multidomain scaffold proteins. (12) Covalent scaffolds are engineered through genetic conjugation of scaffold domains via linkers. Self-assembling scaffolds are generated through covalent coupling of scaffold domains to self-assembling units, such as peptide tags (10)-including leucine zippers, (11) or self-assembling proteins.
Multidomain scaffold proteins (MDSPs) can be divided into two classes: self-assembling or covalent scaffolds.
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