by Huimei Zheng and Stewart N. Loh
Biosensor development continues to be driven by the growing need to detect and monitor analytes in scientific, industrial, environmental and clinical realms. With their well-established capacity for molecular recognition, proteins are the go-to choice of binding elements in many conventional sensor designs. Recently, a new class of biosensors built around protein switches has emerged. Switchable proteins offer the potential of integrating analyte binding and signal transduction within a single molecule, thus reducing the need for complex and expensive detection equipment and opening the door to extreme miniaturisation and in vivo applications.
The past decade has witnessed remarkable advancements in biosensor technology. The annual count of biosensor-related research papers has more than quadrupled over the past ten years. But how imminent is the reality of a generation of sensors that offer accurate and rapid results yet are small, inexpensive, and simple to operate? Among the hundreds (if not thousands) of designs, relatively few—a continuous blood glucose monitor for diabetes, for example—have found widespread use and commercial success. Nonetheless, the scientific community has made remarkable progress towards achieving that goal. Robust research in areas such as new materials, electronics, genetic/protein engineering, and miniaturisation have played vital roles in improving biosensor technology.
A biosensor consists of a binding module and a transducing element with which to convert the binding event into a measurable signal. With their ability to recognise nearly any ligand, biological macromolecules such as proteins (e.g. antibodies) and nucleic acid aptamers make ideal receptors and they fulfill that role in most existing sensors. By contrast there are many technologies for signal transduction. These can be categorised, however, into two designs: (i) electronic devices that detect binding by the difference in optical activity, mass, index of refraction or charge between the free receptor and the receptor-ligand complex (conventional biosensors), and (ii) binding domains that switch conformations upon interaction with the target (protein switches). By combining recognition and transduction functions in a single molecule, protein switches may offer the greatest opportunity for miniaturisation and simplification, as well as the potential for genetic encoding for sensing in vivo.
Detection technologies for conventional biosensors are too varied to enumerate here. Instead we highlight surface plasmon resonance (SPR), the technique that has become a preeminent tool for quantifying binding affinities and rates since its commercialisation in the 1990s. SPR employs a variety of conventional binding domains such as proteins, peptides and nucleic acids. These molecules are attached to a metal surface using one of several coupling chemistries. As the ligand is infused over the immobilised receptor, the surface is interrogated with polarised light fixed at the resonant angle. Binding is detected in real time by the change in the refractive index brought about by the increase in mass density at the liquid/surface boundary.
The major advantages of SPR are primarily that the recognition domain need not undergo any conformational change upon binding, and secondarily that it requires only minimal modification to affix to the sensor surface. The modification typically consists of attaching an affinity tag such as biotin or poly-histidine to the binding domain (ideally at one of its ends to avoid perturbing the structure, folding, or binding site) in order to couple it to an avidin or nickel-decorated sensor chip, respectively. These modest requirements allow a broad assortment of recognition elements to be compatible with SPR. Another attribute of SPR is that it obtains on- and off-rates as well as equilibrium binding constants, and it does so with superior sensitivity over a range of analyte concentrations (extending to nM and below). Innovations in technology continue emerge, particularly in SPR imaging (SPRi), which couples the attractive features of SPR measurements with the capability of visualising the entire chip surface allowing interactions to be monitored continuously and simultaneously in a multi-array fashion .
One of the chief disadvantages of SPR is the requirement for surface adsorption of the binding element. It can be thorny to attach a biological macromolecule to a solid surface without compromising its function. Nonspecific binding of other molecules to the sensor can be significant, especially in impure samples. Another limitation is that the output signal is proportional to the change in mass; consequently, small analytes are considerably more difficult to detect than large ones.
Perhaps the most influential paradigm for a switch-based biosensor is the cameleon calcium sensor [Figure 1], . Cameleon is built around calmodulin (CaM), a protein that undergoes a unique conformational change upon calcium binding. In the presence of Ca2+ CaM adopts a dumb-bell shape in which an extended but flexible alpha helix separates its two metal-binding head domains. This structural change exposes a binding site for a number of regulatory peptides. The two heads wrap tightly around the target peptide, resulting in a compact, globular complex. In cameleon, this transformation is effected by fusing the M13 peptide to the end of CaM, then visualised by placing cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) at the amino and carboxy termini of the protein, respectively. Calcium binding is detected by the increase in Förster resonance energy transfer (FRET) from the CFP donor to the YFP acceptor, as cameleon embraces the M13 peptide and the distance between its termini closes.
Cameleon embodies several of the qualities most desired in a biological sensor. First, it is fully genetically encoded, meaning that it can be introduced into specific locations within cells without cofactors and by genetic means. Second, the use of fluorescence makes detection sensitive as well as amenable to a variety of instruments ranging from simple benchtop fluorimeters to sophisticated single molecule platforms. Finally, the ratiometric nature of FRET ensures that the output response is independent of sensor concentration.
Yet for all of calmodulin’s enabling conformational gyrations there is one overwhelming weakness: nature has provided us with only one of its kind. The great majority of proteins do not change their structures appreciably upon interaction with ligands, and calmodulin’s singular mechanism cannot be readily transferred to other binding proteins. How then can one generate new biosensors for other targets of choice, based on the cameleon archetype?
One solution is to use the tools of protein engineering to introduce a binding-induced conformational change into a protein where none existed previously. A method termed ‘alternate frame folding’ (AFF) was developed recently for this purpose . The unique aspect of AFF is that it links binding to folding of one region of the molecule, and to unfolding of another region. This coupled folding-unfolding reaction constitutes a conformational change that can, in principle, be engineered into many different proteins.
Figure 2 illustrates the steps for converting a generic binding protein into a switch by the AFF design. First, a segment from one of the termini of the protein is duplicated and appended to the opposite end. This segment must contain at least one amino acid which, when changed to another residue, abolishes ligand binding. The duplicated segment is joined to the parent molecule by a peptide linker long enough to span the amino and carboxy termini of the original protein. The twin segment establishes a second ‘frame’ of protein folding. The protein can fold to the wild-type native structure (conformation N) using its normal set of amino acids. It can also fold using the duplicate segment, in which case it adopts what is known as a circularly-permuted structure (see Sidebar). The permuted conformation is also native and functional and is designated N’. The duplicate peptide is anticipated to extend from the amino or carboxy terminus (in N and N’, respectively) as a disordered tail. The protein cannot adopt N and N’ conformations simultaneously because they compete for a shared stretch of amino acids. Accordingly, the protein interconverts between the two forms with the equilibrium distribution determined by their relative thermodynamic stabilities.
The second step is to use site-directed mutagenesis to tune the switch. Point substitutions are introduced in either duplicate region to stabilise N or destabilise N’, so that one is more populated than the other in the absence of ligand. The critical binding residue in the more stable conformation is then mutated to a non-binding amino acid. In this way, the free energy of ligand binding is used to drive the conformational change from N to N’ (or from N’ to N), in which the remodeled binding pocket presents the correct residue for contact with the target.
The final step is to attach fluorescent groups onto the protein to report on the change. A donor is placed at the extreme N-terminus and an acceptor at the position that was chosen for circular permutation. The latter site is usually a surface loop, so these locations are expected to be relatively non-perturbing. The binding-induced conformational change is detected by either an increase or decrease in FRET, as the donor-acceptor distance shortens (N to N’) or lengthens (N’ to N), respectively. Stratton et al. applied the AFF methodology to the small protein calbindin D9k . Calbindin, which corresponds to just one of the Ca2+-binding heads of CaM, does not change structure upon binding. Nevertheless, the AFF modification converted it into a fluorescent calcium sensor similar to cameleon.
The principle advantage that AFF holds over naturally-occuring switches such as cameleon is that AFF is a general technique that can be applied to many proteins. The main limitations of AFF are that the duplicate peptide must not cause the protein to aggregate or be degraded, and the protein must tolerate circular permutation as well as not contain covalent linkages that would physically prevent the fold shift (e.g. disulphide bridges).
What does the future hold for biosensor design? Conventional biosensors will benefit from new and/or increasingly sensitive transduction technologies, microfluidics, new materials and nanoscale fabrication. The potential as well as the limitations of biomolecular switch-based sensors are only just beginning to be explored. In addition to AFF, other switch designs have recently emerged that couple binding to folding  or link various recognition and reporting domains together to engender an open-to-closed conformational change upon binding [5, 6]. The principle challenges to designers of conventional and switch-based sensors alike are to create devices that can operate in real-world, contaminant-filled environments, be miniaturised to the extent that they can work in the human body, and be simplified and economised to the point of widespread commercial availability. The need to detect disease biomarkers, pathogens and environmental threats will continue to fuel demand for new biosensors for the foreseeable future. Within the next decade we anticipate that molecular switches will take their place alongside conventional biosensors in the effort to meet these demands.
This work was supported by N.I.H. grant GM069755 (to S.N.L.).
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Huimei Zheng and Stewart N. Loh*
Department of Biochemistry & Molecular Biology, State University of New York Upstate Medical University,
Syracuse, NY, USA