1. Engineering Biosensors for the Sensitive Detection of Proteases Akshay Sriprasad PI: Dr. Indraneel Ghosh, Department of Chemistry Mentor: Sujan Shekhawat The University of Arizona Arizona Space Grant Consortium Statewide Symposium

2. Proteases Enzymes- Proteins that catalyze reactions. Proteases- Enzymes that cleave peptide (amide) bonds within specific proteins. Proteases implicated in cancer: Caspases: cysteine proteases involved in apoptotic cancer pathways. Human Tissue Kallikriens: regulate cancer cell growth, metastasis and angiogenesis. Image Credit: Peptide Bond. Rafiki Inc. 10 Apr. 2009.www.codefun.com/Images/ Site of cleavage

3. Objective Survival rates for cancer are much higher when it is diagnosed early. Proteases play a role in key cancer pathways (apoptosis). Detection of these pathways can help an early diagnosis. Objective: Detect specific protease activity in an in-vitro state using biosensors.

4. Biosensors What is a biosensor? A device that monitors and transmits information about a life process. 1 Three components in our specific biosensor. Interacting component: responds to a specific reaction or interaction. Reporter component: converts response from interacting elements into a measurable physiochemical signal. Signal processing component: instrument that displays data in a meaningful quantitative manner. 1 "Biosensor." Merriam-Webster.

5. Split-Protein Reporter Approach Basis A and B are two initially inactive protein fragments. When tethered domains interact, A and B reassemble, producing signal. Interacting Component: ID1 and ID2 are two complementary proteins that come together spontaneously in solution Reporter Component: Upon complementation of ID1 and ID2, A and B which are tethered to ID1 and ID2, respectively, are brought together and emit signal Image courtesy of Sujan Shekhawat, Ghosh group

6. Split-Protein Reporter Approach A & B Two halves of Firefly Luciferase. Named N-Fluc and C-Fluc, for the N and C termini of proteins. Protein that emits bioluminescence in fireflies; signal in this experiment in the form of luminescence ID1 & ID2 Coiled Coils “A coiled coil is a bundle of α-helices that are wound into a superhelix” 1 Heptad: A repeat of seven amino acids (residues), with two out of every seven being hydrophobic. During complementation, hydrophobic residues wrap around each other in the middle, being surrounded by polar residues and the aqueous medium 1. A. Lupas, TIBS, 1996, 21, 375-382 Image courtesy of Sujan Shekhawat, Ghosh group Image courtesy of Jenny Furman, Ghosh group

7. The Experiment Basis: Inhibit one coiled coil in the biosensor with its partner, bound by a protease cleavable linker. Upon addition of protease, inhibiter is detached and two halves of system may interact. Experiment has been performed for designed coiled coil pair “Acid” and “Base.” Shekhawat et al, Angew. Chem. Int. Ed. Manuscript submitted

8. My Investigation: EE- RR EE and RR are another pair of coiled coils. Higher affinity for each other than Acid and Base. Orientation preference allows for optimization. I mutated the inhibitor coiled coil residues to alter inhibition/free states. Those corresponding to hydrophobic interactions disrupted (L to A). Weakens inhibitor in free state. One residue corresponding to parallel preference disrupted (N to A). Strengthens inhibition for lower background.

9. My Investigation: EE- RR Performed two different tests with varying degree of destabilization Two L-A vs control S/N: 5.4x to 4.4x Two L-A vs Five L-A S/N: 2.1x to 4.5x

10. Conclusions EE/RR emits much higher luminescence but S/N ratio is not quite as high as Acid/Base pair. For EE/RR system, 5 hydrophobic destabilizations gives a higher S/N ratio than 2 destabilizations, which gives a higher S/N than no destabilizations. Mutation theory produces hypothesized results.

11. Future Directions Acid/Base system has successfully been applied to Caspase-3 protease, central to cell apoptosis. System undergoing application to five other human disease proteases. Testing inside cells. http://www.emdbiosciences.com/html/cbc/apoptosis_Roll.html

12. Thank You