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Nature's reliance on proteins to carry out nearly all biological processes has led to the evolution of biomolecules that exhibit a seemingly endless range of functions. Much research has been devoted toward advancing this process in the laboratory in order to create new proteins with improved or unique capabilities. The protein-engineering field has rapidly evolved from pioneering studies in engineering protein stability and activity to an application-driven powerhouse on the forefront of emerging technologies in biomedical engineering and biotechnology. A classic protein-engineering technique in the medical field has focused on manipulating antibodies and antibody fragments for various applications. New classes of alternative scaffolds have recently challenged this paradigm, and these structures have been successfully engineered for applications including targeted cancer therapy, regulated drug delivery, in vivo imaging, and a host of others. This review aims to capture recent advances in the engineering of nonimmunoglobulin scaffolds as well as some of the applications for these molecular recognition elements in the biomedical field.

A better understanding of the conformational changes exhibited by intrinsically disordered proteins is necessary as we continue to unravel their myriad biological functions. In repeats in toxin (RTX) domains, calcium binding triggers the natively unstructured domain to adopt a beta roll structure. Here we present an in vitro Forster resonance energy transfer (FRET)-based method for the investigation of the conformational behavior of an RTX domain from the Bordetella pertussis adenylate cyclase consisting of nine repeat units. Equilibrium and stopped-flow FRET between fluorescent proteins, attached to the termini of the domain, were measured in an analysis of the end-to-end distance changes in the RTX domain. The method was complemented with circular dichroism spectroscopy, tryptophan fluorescence, and bis-ANS dye binding. High ionic strength was observed to decrease the calcium affinity of the RTX domain. A truncation and single amino acid mutations yielded insights into the structural determinants of beta roll formation. Mutating the conserved Asp residue in one of the nine repeats significantly reduced the affinity of the domains for calcium ions. Removal of the sequences flanking the repeat domain prevented folding, but replacing them with fluorescent proteins restored the conformational behavior, suggesting an entropic stabilization. The FRET-based method is a useful technique that complements other low-resolution techniques for investigating the dynamic conformational behavior of the RTX domain and other intrinsically disordered protein domains.

Typically, chromatography is the most costly and time-consuming step in protein purification. As a result, alternative methods have been sought for bioseparations, including the use of stimulus-responsive tags that can reversibly precipitate out of solution in response to the appropriate stimulus. While effective, stimulus-responsive tags tend to require temperature changes or relatively harsh buffer conditions to induce precipitation. Here we describe a synthetic peptide, based on the natural repeat-in-toxin (RTX) domain that undergoes gentler calcium-responsive, reversible precipitation. When coupled to the maltose binding protein (MBP), our calcium-responsive tag efficiently purified the fusion protein. Furthermore, when the MBP was appended to green fluorescent protein (GFP), β-lactamase, or a thermostable alcohol dehydrogenase (AdhD), these constructs could also be purified by calcium-induced precipitation. Finally, protease cleavage of the precipitating tag enables the recovery of pure and active target protein by cycling precipitation before and after cleavage.

The use of repetitive peptide sequences forming predictable secondary structures has been a key paradigm in recent efforts to engineer biomolecular recognition. The modularity and predictability of these scaffolds enables precise identification and mutation of the active interface, providing a level of control which non-repetitive scaffolds often lack. However, the majority of these scaffolds are well-folded stable structures. If the structures had a stimulus-responsive character, this would enable the allosteric regulation of their function. The calcium-responsive beta roll-forming repeats in toxin (RTX) domain potentially offer both of these properties. To further develop this scaffold, we synthesized a set of RTX peptides ranging in size from 5 to 17 repeats, with and without C-terminal capping. We found that while the number of repeats can be altered to tune the size of the RTX face, repeat ordering and C-terminal capping are critical for successful folding. Comparing all of the constructs, we also observed that native configuration with nine repeats exhibited the highest affinity for calcium. In addition, we performed a comparison on a set of known RTX-containing proteins and find that C-terminal repeats often possess deviations from the consensus RTX sequence which may be essential for proper folding. We further find that there seems to be a narrow size range in which RTX domains exist. These results demonstrate that the deviations from the consensus RTX sequence that are observed in natural proteins are important for high-affinity calcium binding and folding. Therefore, the RTX scaffolds will be less modular as compared with other, non-responsive scaffolds, and the sequence-dependent interactions between different repeats will need to be retained in these scaffolds as they are developed in future protein-engineering efforts.

Intrinsically disordered peptides (IDPs) have recently garnered much interest because of their role in biological processes such as molecular recognition and their ability to undergo stimulus-responsive conformational changes. The block V repeat-in-toxin motif of the Bordetella pertussis adenylate cyclase is an example of an IDP that undergoes a transition from a disordered state to an ordered beta roll conformation in the presence of calcium ions. In solution, a C-terminal capping domain is necessary for this transition to occur. To further explore the conformational behavior and folding requirements of this IDP, we have cysteine modified three previously characterized constructs, allowing for attachment to the gold surface of a quartz crystal microbalance (QCM). We demonstrate that, while immobilized, the C-terminally capped peptide exhibits similar calcium-binding properties to what have been observed in solution. In addition, immobilization on the solid surface appears to enable calcium-responsiveness in the uncapped peptides, in contrast to the behavior observed in solution. This work demonstrates the power of QCM as a tool to study the conformational changes of IDPs immobilized on surfaces and has implications for a range of potential applications where IDPs may be engineered and used including protein purification, biosensors, and other bionanotechnology applications.

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