This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z-domain of the chimera (Z-PYP-AA) showing >30-fold lower dark-state binding and no loss in switching. A thermodynamic model indicated that mutations to decrease Z-domain folding energy would alter target affinity without loss of switching. Blue light caused loss of structure in PYP and a 2-6-fold change in the apparent affinity of Z-PYP for its target as determined using size exclusion chromatography, UV–Vis based assays, and ELISA. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z-domain was unfolded. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. ![]() Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. This thesis additionally describes the development and characterization of a synthetic protein that was designed to investigate chromophore reconstitution in photoactive yellow protein (PYP), a promising scaffold for engineering photo-controlled protein tools. In addition, a tunable selection system is presented, which allows for the targeted selection of protein-protein interactions of a desired affinity range. ![]() A screening system is described, which permits detection of DNA-binding activity based on conditional expression of a fluorescent protein. This thesis describes the development of two genetic circuits that can be used to evaluate libraries of switchable proteins, enabling optimization of both the on- and off-states. Directed evolution, however, has been relatively infrequently used to develop photo-switchable proteins due to the challenge presented by high-throughput evaluation of switchable protein activity. Complementing this approach with directed evolution would be expected to facilitate these efforts. The development of novel photo-switchable tools has to date largely relied on rational design. The success of this approach has fueled the development of tailored photo-switchable proteins, to enable targeted molecular events to be studied using light. Light-switchable proteins are being used increasingly to understand and manipulate complex molecular systems. M121E-cPYP thus provides a scaffold that may allow a wider range of photoswitchable protein designs by replacing the linker polypeptide with a target protein or peptide sequence. Fluorine NMR studies with fluoro-tryptophan labeled M121E-cPYP show that blue-light drives large changes in conformational dynamics and leads to solvent exposure of Trp7 (Trp119 in wtPYP numbering) consistent with substantial rearrangement of the N-terminal cap structure. NMR, circular dichroism, and UV-Vis analysis indicated that the M121E-cPYP mutant also adopts a dark state structure like that of wtPYP although protonated and deprotonated forms of the chromophore coexist giving rise to a shoulder near 380 nm in the UV-Vis absorption spectrum. Targeted mutations at M121E (M100 in wtPYP numbering) were found to enhance the light sensitivity substantially by lengthening the lifetime of the light state to ~10 min. ![]() However, thermal recovery of dark state cPYP is ~10-fold faster than wtPYP, so that very bright light is required to significantly populate the light state. Biophysical analysis indicated that this cPYP adopts a dark state conformation much like wtPYP and undergoes wtPYP-like photoisomerization driven by blue light. We created a test cPYP by connecting the N- and C- termini of wild type PYP (wtPYP) with a GGSGGSGG linker polypeptide and introducing new N- and C- termini at G115 and S114 respectively. For such a design strategy to succeed, the circularly permuted PYP (cPYP) would have to fold normally and undergo a photocycle similar to that of the wild type protein. We reasoned that this conformational change might be used to control other protein or peptide sequences if these were introduced as linkers connecting the N and C-termini of PYP in a circular permutant. Upon blue light irradiation, photoactive yellow protein (PYP) undergoes a conformational change that involves large movements at the N-terminus of the protein.
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