Engineering Protein Switches: Sensors, Regulators, and Spare Parts for Biology and Biotechnology

Golynskiy, M Misha; Koay, Mst Melissa; Vinkenborg, JL Jan; Merkx, Maarten

doi:10.1002/cbic.201000642

Cited by 42 publications

(36 citation statements)

References 106 publications

(119 reference statements)

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“…Such a conformational switch protein has great advantages in cell signaling, because it can be used as a universal regulatory domain (9) for precise, specific, and temporal control over rapidly activated signaling proteins (5,(10)(11)(12)(13)(14)(15). Traditional genetically encoded methods for temporal protein control at the protein level have several drawbacks (5,13).…”

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confidence: 99%

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Rational design of a ligand-controlled protein conformational switch

Dağliyan

Shirvanyants

Karginov

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

114

115

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Design of a regulatable multistate protein is a challenge for protein engineering. Here we design a protein with a unique topology, called uniRapR, whose conformation is controlled by the binding of a small molecule. We confirm switching and control ability of uniRapR in silico, in vitro, and in vivo. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two unique phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell-cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.spatiotemporal control | cell motility | endothelial-mesenchymal transition T he past two decades have seen a revolution in computational protein design, with remarkable milestones including design of a helical protein from first principles (1), redesign of zinc finger proteins (2), and de novo design of an α/β protein (3). These studies highlighted, as a proof of principle, our ability to rationally control the structure of proteins by using basic physical principles and phenomenology. These approaches are based on finding an optimal sequence for a given single structure or ensemble of related states, and do not provide a strategy to construct a protein capable of large on-demand conformational transitions (4, 5). A number of multistate protein design algorithms (4, 6) have been proposed; however, designing an experimentally confirmed, regulatable multistate protein, or a conformational switch (5), still remains as a challenging task because of the necessity of engineering and controlling multiple protein states (4,7,8).Such a conformational switch protein has great advantages in cell signaling, because it can be used as a universal regulatory domain (9) for precise, specific, and temporal control over rapidly activated signaling proteins (5, 10-15). Traditional genetically encoded methods for temporal protein control at the protein level have several drawbacks (5, 13). Recently developed protein switches, including derivatives of the light, oxygen, or voltage (LOV) domain (16, 17), can provide direct control at the protein level with light, but cannot be readily used in nontransparent animals. Our previous rapamycin regulated (RapR) kinase method (14) can potentially overcome this problem, but it requires expression and control of two proteins. The variable stoichiometry of these proteins renders the response more heterogeneous and essentially impractical in animals. Therefore, a single-chain, insertable, and transferable regulatory domain would be very valuable.Here we design a ligand-controlled conformational switch, uniRapR, a potentially broadly applicable, single-chai...

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confidence: 99%

“…Traditional genetically encoded methods for temporal protein control at the protein level have several drawbacks (5,13). Recently developed protein switches, including derivatives of the light, oxygen, or voltage (LOV) domain (16,17), can provide direct control at the protein level with light, but cannot be readily used in nontransparent animals.…”

mentioning

confidence: 99%

Rational design of a ligand-controlled protein conformational switch

Dağliyan

Shirvanyants

Karginov

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

114

115

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“…Through conformational transitions, these proteins can modulate their interactions with other proteins or ligands to perform important biological functions such as cellular signaling and targeted drug delivery. [8][9][10] This ambiguous fold propensity of proteins may have profound implications in understanding protein evolution, mutation related diseases, and functional annotation of protein sequences of unknown structure. Thus, successful design of such proteins may help to achieve novel functionalities used for biological imaging, biosensors, 11 and therapeutic agents 12 that requires large scale on demand conformational rearrangement.…”

Section: Introductionmentioning

confidence: 99%

Designing pH induced fold switch in proteins

Baruah

Biswas

2015

The Journal of Chemical Physics

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This work investigates the computational design of a pH induced protein fold switch based on a self-consistent mean-field approach by identifying the ensemble averaged characteristics of sequences that encode a fold switch. The primary challenge to balance the alternative sets of interactions present in both target structures is overcome by simultaneously optimizing two foldability criteria corresponding to two target structures. The change in pH is modeled by altering the residual charge on the amino acids. The energy landscape of the fold switch protein is found to be double funneled. The fold switch sequences stabilize the interactions of the sites with similar relative surface accessibility in both target structures. Fold switch sequences have low sequence complexity and hence lower sequence entropy. The pH induced fold switch is mediated by attractive electrostatic interactions rather than hydrophobic-hydrophobic contacts. This study may provide valuable insights to the design of fold switch proteins.

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“…In that sense, photoreceptors could be useful candidates as light-responsive units to control activity of biomolecules. 2 PYP known as a photosensor protein has been isolated from Ectothiorhodospira halophila. 3 Exposure of PYP to visible light ( = 446 nm) causes partial unfolding of -helices at N-terminal region.…”

Section: Introductionmentioning

confidence: 99%