Fluorescent protein biosensors have unlocked major advances in cell biology, neurobiology, clinical chemistry, environmental monitoring, and agricultural technologies¹.  The best-in-class fluorescent sensors transduce chemical events into robust fluorescent signals.  Binding of a small molecule to the sensor (e.g. glucose or neurotransmitters), interactions of a sensor with another protein (e.g. cellular signal transduction networks), or changes in the physical environment of the sensor such as a strong electrical field (e.g. detection of electrochemical pulses along a neuron) are typical chemical events.  Biosensor development depends on a deep understanding of molecular mechanisms that transduce the chemical reactions (events) into a fluorescent signal, which can be challenging to establish.  In the accompanying article², we have uncovered a subtle, but powerful, fluorescent signal transduction mechanism (Figure 1) that we anticipate will enable development of new fluorescent biosensors.

Figure 1.  A conformationally sensitive fluorescent color-switching mechanism.  Thiol-reactive Prodan derivatives (top left) can be site-specifically coupled to proteins via cysteine residues.  Prodan comprises a two-membered aromatic ring with dimethyl amine and carbonyl-containing groups, which can twist independently relative to the plane of the two rings.  X-ray structures revealed that in protein conjugates, the carbonyl can be twisted by specific interactions with two or three amino acid side-chains.  In the planar form, the corresponding fluorescent emission is green, whereas the twisted fluorophore emits with a blue color.  We demonstrated this effect in glucose-binding proteins that adopt distinct conformations in response to glucose.  The apo- and glucose-bound protein conformations are coupled to different fluorophore twists, creating a two-color glucose sensor.

At the heart of any fluorescent signal transduction mechanism is the phenomenon of conformational coupling which Jacques Monod referred to as the “secret of life”, because it constitutes the molecular basis of most biological processes such as enzyme control, ligand transport across membrane, motor proteins, or cellular signal transduction³.   In conformational coupling, ligand binding or perturbations of the physical environment are associated with changes in the distribution of protein conformational states.  Accordingly, fluorescent biosensors are created by coupling distinct protein conformations to changes in the electronic structure of a fluorophore, or pairs of fluorophores that have been engineered into the protein.

Many fluorescent biosensors exploit conformation-dependent changes in the fluorescence resonance energy transfer (FRET) between two engineered fluorophores that emit at two different colors (wavelength ranges).  A change in the distance between appropriately chosen fluorophore pairs alters their relative emission intensities.  Even though the FRET distance dependence is steep (the sixth power of inter-fluorophore separation), it is challenging to engineer FRET sensors with large signal changes if the protein conformational changes of interest are small or confined to small regions within the protein, as is the case for many conformational coupling events.  Consequently, different classes of fluorophores whose response mechanisms directly probe delicate local structural changes could be more generalizable tools to build fluorescent biosensors.

A family of naphthalene-based fluorophores, the Prodan family, was developed in the early 1970s by Gregorio Weber’s research group who argued that pairing an electron donor group (amine) with an acceptor (carbonyl) on either end of a two-membered aromatic ring system would result in a fluorescent excited state that is particularly sensitive to electrostatic environment of the fluorophore⁴.  Attachment of Prodan derivatives at specific sites in proteins resulted fluorescent biosensors that successfully monitored ligand-mediated conformational changes.  Remarkably, these single-fluorophore conjugates generated signals that switched between two different colors, albeit with varying degrees of color shifts.  Following the original design hypothesis, these color changes were interpreted as arising from solvatochromic effects in which protein conformational changes altered the solvent structure surrounding the attached fluorophore, thereby affecting electric fields that influence the energy levels of the excited state, and hence their fluorescence emission colors.  Like the distance-dependent effects in FRET, solvatochromism does not probe local conformations directly, and does not provide clear design principles for creating new biosensors.

However, as detailed in the accompanying paper, we discovered that the Prodan color-switching mechanism actually involves specific interactions between the fluorophore carbonyl and a small number of protein amino acid side-chains.  These localized interactions change in response to ligand-mediated local protein conformational changes.  This conformational coupling mechanism therefore can probe delicate structural changes, and is amenable to structure-based computational design approaches, as is desired for a generalizable biosensor engineering principle.

Figure 2.  Glucose-mediated conformational changes of E. coli glucose-binding protein.  In the absence of glucose, the protein adopts an open conformation (left).  Binding of glucose shifts the population to a closed conformation in which glucose is enveloped by surfaces contributed by both domains (right).

Using spectral analysis tools, X-ray crystallography, and mutagenesis of periplasmic bacterial glucose-binding proteins (GBPs), we showed that the thiol-reactive Prodan derivatives Acrylodan and Badan switch between predominantly blue and green colors in response to glucose binding.²  GBPs comprise two domains connected by a hinge.  Glucose binds between these two domains.  The binding reaction provokes a conformational change from an open, apo-protein, conformation in which the interdomain interface surfaces are apart from each other, and a closed conformation in which the surfaces are close together, enveloping the bound glucose.  Fluorophores attached at different positions can evoke either a blue®green, or a green®blue color switch.  X-ray crystallography revealed that the green color was correlated with fluorophore conformation that was planar, and the blue color with a conformation in which the carbonyl was twisted by ~30° out of plane. In both cases the carbonyl was held in place by grasping the naphthalene ring in a channel between the two domains, and the carbonyl by specific interactions with two or three amino acid side-chains.  These interactions are unique to the fluorophore attachment positions, enabling color switching in either direction, depending on steric details of the interactions between protein and fluorophore.  This mechanism therefore directly probes local protein conformational, and is amenable to structure-based fluorescent biosensor design strategies by computing the interactions between fluorophore conformations and protein residues.

Mutagenesis studies revealed a further subtility: the apo-proteins and glucose complexes can adopt a range of colors intermediate between pure blue and green forms.  This phenomenon established that the conjugated fluorophores can adopt a mixture of planar and twisted conformations within a given protein conformation.  Pure colors are formed in single-membered ensembles of planar or twisted fluorophores; ensembles comprising mixtures of both conformations give rise to intermediate colors.  Each distribution is determined by the detailed interactions between the twistable carbonyl and the protein amino acid side-chains.  This mechanism guides fluorescent sensor signal optimization: introduce mutations that bias fluorophore twisting within a protein conformation.

We uncovered this conformational coupling mechanism in fluorescent glucose sensor proteins, which have been used to construct continuous monitoring systems for the management of diabetes.  The same principles can be extended to other proteins to create a large variety of fluorescent biosensors.

References

1.  Demchenko, A. P. Introduction to Fluorescence Sensing. Third edn,  (Springer, 2020).

2. Allert, M. J., Kumar, S., Wang, Y., Beese, L. S. & Hellinga, H. W. Chromophore carbonyl twisting in fluorescent biosensors encodes direct readout of protein conformations with multicolor switching. Nature Communications Chemistry 168 (2023).

3. Phillips, R. The Molecular Switch -Signaling and Allostery.  (Princeton University Press, 2020).

4. Valeur, B., and Leray, I. Design principles of fluorescent molecular sensors for cation recognition. Coordination Chemistry Reviews 205, 3-40 (2000).