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. 2012 Aug;21(8):1222-30.
doi: 10.1002/pro.2109.

Designing redox potential-controlled protein switches based on mutually exclusive proteins

Qing Peng  1 , Na Kong, Hui-Chuan Eileen Wang, Hongbin Li
Affiliations

Designing redox potential-controlled protein switches based on mutually exclusive proteins

Qing Peng et al. Protein Sci. 2012 Aug.

Abstract

Synthetic/artificial protein switches provide an efficient means of controlling protein functions using chemical signals and stimuli. Mutually exclusive proteins, in which only the host or guest domain can remain folded at a given time owing to conformational strain, have been used to engineer novel protein switches that can switch enzymatic functions on and off in response to ligand binding. To further explore the potential of mutually exclusive proteins as protein switches and sensors, we report here a new redox-based approach to engineer a mutually exclusive folding-based protein switch. By introducing a disulfide bond into the host domain of a mutually exclusive protein, we demonstrate that it is feasible to use redox potential to switch the host domain between its folded and unfolded conformations via the mutually exclusive folding mechanism, and thus switching the functionality of the host domain on and off. Our study opens a new and potentially general avenue that uses mutually exclusive proteins to design novel switches able to control the function of a variety of proteins.

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Figures

Figure 1
Figure 1
Design principle of the redox potential-dependent protein switch based on a mutually exclusive protein design. (A) Three-dimensional structure of GB1 (PDB code: 1PGB). The hFc binding epitope of GB1 is highlighted in yellow. Residues that are in the hFc binding epitope of GB1 and in direct contact with hFc are highlighted in yellow. (B) The three-dimensional structure of the GC2/hFc complex (PDB code: 1FCC). GC2 is highly homologous to GB1, only differing by two residues. The structure of GC2/hFc has been used as a model for the GB1/hFc complex. GC2 is colored in green and hFc is colored in yellow. In (A and B), the loop where I27w34f is inserted to construct the mutually exclusive protein is highlighted in blue. (C) The schematic showing the equilibrium of the conformational change between the oxidized form and the reduced form of the mutually exclusive protein GL5CC–I27w34f. The equilibrium can be controlled using either oxidizing and reducing reagents. GL5CC is colored in green, and I27w34f is colored in pink. The two cysteine residues are colored in yellow. In the oxidized conformation GL5CC(F)–I27w34f(U), the mutually exclusive protein should have a high binding affinity to hFc; in the reduced state GL5CC(U)–I27234f(F), the mutually exclusive protein should not bind to hFc.
Figure 2
Figure 2
Typical tryptophan fluorescence traces showing the folding of GL5-I27w34f in 0.3M GdmCl. Black trace is in the absence of hFc and gray trace is in the presence of 1.75 mg/mL hFc. The presence of hFc increased the relative population of GL5(F)–I27w34f(U), but GL5(U)–I27w34f(F) is still the dominant conformation of the mutually exclusive protein, suggesting that I27w34f is thermodynamically more stable than the GL5–hFc complex.
Figure 3
Figure 3
Typical tryptophan fluorescence traces for the folding of GL5CC–I27w34f in 0.3M GdmCl. The gray curve represents the oxidized form, where the black curve is from the reduced form. The tryptophan fluorescence of GL5CC–I27w34f exhibits a fast rising phase followed by a much slower decay phase. The amplitude of the decay phase in the oxidized mutually exclusive protein is significantly smaller than that in the reduced conformation. In the oxidized mutually exclusive protein, a double exponential fit to the folding phase of GL5 domain results in rate constants of 3.9 and 22.6 s−1 in 0.3M GdmCl (gray). The unfolding phase of GL5 is best described by a double exponential fit with rate constants of 0.087 and 0.022 s−1 in 0.3M GdmCl (gray). In the reduced mutually exclusive protein, the folding phase of the GL5 domain can be well-described by a single exponential with a rate constants of 7.8 s−1 (black). At the same time, the decay phase is best described by a double exponential fit with rate constants of 0.30 and 0.039 s−1, respectively (black).
Figure 4
Figure 4
Far UV CD spectra of the mutually exclusive protein show that the reduction of the disulfide can convert GL5CC(F)–I27w34f(U) to GL5CC(U)–I27w34f(F). (A) Far UV CD spectra of GL5CC–I27w34f in the oxidized and reduced condition. (B) Time evolution of the ellipticity at 221 and 230 nm of the mutually exclusive protein after addition of 6 mM DTT.
Figure 5
Figure 5
SPR sensorgrams for the binding and dissociation of oxidized (A) and reduced (B) forms of mutually exclusive protein GL5CC–I27w34f to hFc. In SPR experiments, hFc was immobilized onto the SPR chip. Concentrations of analytes are indicated on each individual curve. Using the Langmuir 1:1 association model, the dissociation constant Kd for the oxidized form of GL5CC–I27w34f to hFc was determined to be 233 nM. In contrast, the reduced form of GL5CC–I27w34f does not show significant binding to hFc.
Figure 6
Figure 6
SPR sensorgrams tracking the binding and dissociation of oxidized (A) and reduced (B) GL15CC to hFc. Analyte concentration is indicated on individual curves. Using the Langmuir 1:1 association model, the dissociation constants of hFc were determined to be 184 nM for the oxidized GL15CC, and 371 nM for the reduced GL15CC.
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