1

DEVELOPING A FRET-BASED ATP BIOSENSOR THROUGH DIRECTED EVOLUTION

By Henry Do

INTRODUCTION:

Adenosine 5’-triphosphate (ATP) is the major energy currency of cells and drives the majority of biochemical reactions within the cell. However, there are few methods for monitoring ATP levels inside individual cells in real-time. A common real-time monitoring

method is the use of chemiluminescence by luciferase, but it presents several problems; for example, in chemiluminescence by luciferase depends not only on the intracellular ATP level but

also on the luciferase concentration, as well as the other substrates, oxygen and luciferin. Moreover, pH also affects luciferase activity 1. Another drawback of this method is that the intracellular ATP level could be perturbed because of ATP consumption. Conventional ATP

quantification methods can only provide the averaged ATP level of an ensemble of cells based on cell extract analysis. Moreover, the distribution pattern of ATP between different intracellular

compartments is unclear 1. A recent method has also been reported to visualize ATP levels in real-time at the single

cell level generates a series of FRET-based indicators for ATP that are composed of the Ɛ

subunits of the bacterial 𝐹0𝐹1-ATP synthase2 sandwiched by the cyan- and yellow-fluorescent

proteins.3. The Ɛ subunits of the bacterial 𝐹0𝐹1 -ATP synthase confers several advantage for a FRET-based ATP biosensor. One is that the Ɛ subunit binds to ATP, but does not hydrolyze it.

Another advantageous feature of the Ɛ subunit is the very high specificity for ATP over other nucleotides; ADP, GTP, CTP, and UTP 4.

However, they utilized rational protein design methods – such as site directed and point mutations - to alter key residues in order to produce higher FRET signal-to-noise ratios. The disadvantage of this method it is still unclear what exactly determines the difference in affinity

between two the Ɛ subunits derived from this technique. This biosensor design will use a directed evolution technique called In-Vitro

Compartmentalization (IVC), an alternative to rational protein design, and selective for Ɛ subunits with higher reversible binding affinity and FRET signal-to-noise ratio. This method deemphasizes the need to understand the mechanisms for binding affinity, but instead employs

and iterative process that produces Ɛ subunits proteins with activities and functions of interest through the process of natural selection.

OVERVIEW: TECHNOLOGY AND TECHNIQUES

Forster resonance energy transfer (FRET) is a technique that allows us to study dynamic protein interactions. When combined with multiple, colored fluorescent proteins, FRET permits high spatial resolution assays of protein-protein interactions in living cells. However, because

FRET signals are usually small, their measurement requires careful interpretation and several control experiments 5 6.

Fluorescence Activated Cell Sorter (FACS) is a device that provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based

2

upon the specific light scattering and fluorescent characteristics of each cell. Modern FACS machines can routinely analyze and sort >107 events per hour, and fluorescence is a sensitive

and widely applicable signal. Compartmentalization of single cells in emulsion droplets also

provides unusually high enzyme concentration, thus enabling detection at low signal-to-noise ratios 7.

In Vitro Compartmentalization (IVC) is a directed evolution technique for selecting

proteins and peptides for binding. The general strategy of the IVC technique is to partition reactions in water droplets dispersed in water-oil emulsions. The cell-like volumes of these

compartments (as low as one femtolitre), and the ability to freely determine and regular their content, and the large number of compartments (>1010 per milliliter emulsion) 8 makes it a

suitable method for optimizing the binding affinity of the Ɛ subunit to ATP without hydrolyzing it. Compartmentalization of genes in emulsion serves as a way of establishing a physical linkage

between the gene and the protein it encodes. The expressed protein is coupled covalently or noncovalently to the gene that encodes it, within the emulsion droplet, either directly or using

microbeads. The emulsion is then broken, and the gene-protein complexes recovered; those with the desired activity are enriched by affinity purification using the target ligand or by FACS of the gene-bead-protein-ligand complexes.

DEVICE

My proposed biosensor will allow bioengineers to monitor real-time the energy usage of their cells with a higher signal-to-noise ratio than current methods. It will also enhance current capacities to tune ATP-dependent biomolecular activity through careful modulation of

extracellular and intracellular pH, which affect affects the proton gradient driving ATP production. Evolution is economical: related enzymes that catalyze different reactions share conserved functional elements, including catalytic or metal-binding residues, cofactors, and key

residues that modulate the chemistry 9. Since designing enzymes is still beyond out capabilities for anything but the simplest reactions, it makes sense to borrow inspiration from the

evolutionary mechanisms nature has devised and exploited. 10 Figure 1: Schematic representation of IVC: In Step 1, an in vitro transcription or translation reaction mixture containing a library of genes linked to a substrate for the reaction being selected is dispersed to form aqueous compartments (in a water-in-oil emulsion) containing typically one gene. The genes are transcribed and translated within their compartments. Our system will encapsulate the Ɛ subunits of the bacterial 𝐹0𝐹1-ATP synthase (Step 2). Subsequently (Step 3), Ɛ subunit proteins with enzymatic activities convert the substrate into a product that remains linked to the gene. Compartmentalization prevents the modification of genes in other compartments. Next (Step 4), the emulsion is broken, all reactions are stopped and the aqueous compartments combined. Genes linked to the product are selectively enriched using an antibody that binds the product but not the substrate), and are then amplified, and either characterized with FACS (Step 5), or linked to the substrate & compartmentalized for further rounds of selection (Step 6). 8

3

Biorecognition - Development of a Series of ATP Indicators .

The higher-affinity Ɛ subunit gene of the bacterial 𝐹0𝐹1-ATP synthase will be derived from

Bacillus subtilis. The genes are transcribed and translated within their compartments. Our system will encapsulate the Ɛ subunits of the bacterial 𝐹0𝐹1-ATP synthase. Figure 1 details the process

of producing higher-affinity Ɛ subunits. We will select for the highest FRET-intensity gene-linked Ɛ subunits and repeat this process iteratively.

Transducer – ATP Binding to Ɛ subunits Induces a Conformational Change.

Once the gene for the desired ε subunit has been determined and isolated, we’ll genetically link mseCFP - a variant of cyan fluorescent protein (CFP) - with monomeric (A206K) Venus

(mVenus) - a variant of yellow fluorescent protein (YFP) - each at either the N- or C terminus of the ε subunit, which is derived from Bacillus subtilis FoF1-ATP synthase. The Ɛ subunits undergoes a large conformational change into a folded form upon ATP binding, by bundling the

two α-helices, which are relaxed in the absence of ATP. This large conformational change upon ATP binding produces a FRET signal since it’s distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor

molecule to an acceptor molecule without emission of a photon.

Readout – FRET Signal

As shown in figure 2, ATP binding to the ε subunit will produce a larger magnitude FRET

signal, which can be measured through a fluorescence microscope by using a 435nm laser on a sample containing Ɛ-mseCFP-mVenus for it to emit a wavelength of 527, that can be used to

study ATP in real-time

Figure 2: FRET-based ATP Ɛ subunit.

Schematic drawing showing the ATP-free

form (left) extended and flexible

conformations of the subunit separate the

two fluorescent proteins, resulting in low

FRET efficiency. In the ATP-bound form

(right), the subunit retracts to draw the two

fluorescent proteins close to each other,

which increase FRET efficiency and signal,

as indicated by the larger arrow. 3

4

BIBLIOGRAPHY

1. Kennedy, H. J. et al. Glucose generates sub-plasma membrane ATP microdomains in single islet ??-cells. Potential role for strategically located mitochondria. J. Biol. Chem. 274, 13281–13291 (1999).

2. von Ballmoos, C., Wiedenmann, A. & Dimroth, P. Essentials for ATP Synthesis by F 1 F 0 ATP Synthases. Annu. Rev. Biochem. 78, 649–672 (2009).

3. Imamura, H. et al. Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Proc. Natl. Acad. Sci. U. S. A. 106, 15651–15656 (2009).

4. Kato-Yamada, Y. & Yoshida, M. Isolated ε subunit of thermophilic F1-ATPase binds ATP. J. Biol. Chem. 278, 36013–36016 (2003).

5. Piston, D. W. & Kremers, G.-J. Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem. Sci. 32, 407–414 (2007).

6. Marvin, J. S., Schreiter, E. R., Echevarría, I. M. & Looger, L. L. A genetically encoded, high-signal-to-noise maltose sensor. Proteins Struct. Funct. Bioinforma. 79, 3025–3036 (2011).

7. Miller, O. J. et al. Directed evolution by in vitro compartmentalization. Nat. Methods 3, 561–570 (2006).

8. Griffiths, A. D. & Tawfik, D. S. Miniaturising the laboratory in emulsion droplets. Trends Biotechnol. 24, 395–402 (2006).

9. Bartlett, G. J., Borkakoti, N. & Thornton, J. M. Catalysing new reactions during evolution: economy of residues and mechanism. J Mol Biol 331, 829–60 (2003).

10. Arnold, F. H. The nature of chemical innovation : new enzymes by evolution *. (2015). doi:10.1017/S003358351500013X