Engineering Biosensors for the

Sensitive Detection of Proteases

Akshay SriprasadPI: Dr. Indraneel Ghosh, Department of

ChemistryMentor: Sujan ShekhawatThe University of Arizona

Arizona Space Grant ConsortiumStatewide Symposium

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/ Genetic/tRNA/image004.jpg>.

Site of cleavage

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.

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. <http://www.merriam-webster.com/dictionary/biosensor>

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

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

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

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.

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

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.

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

Thank You