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A genetically programmable protein module as intracellularly deliverable QD-based FRET probes for viral protease detection

Nikola Finneran, Divya Sivaraman, Payal Biswas, and Wilfred ChenDepartment of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521

Abstract

Proteases are enzymes that are used in various cellular processes such as blood coagulation, hormone maturation and apoptosis. They are also used as the key virulence factor for pathogenic infection. These properties make proteases a prime target for detailed investigation to better understand the disease development process and can be potentially used to study various therapeutic agents. One of the most promising methods for probing protease activity is based on the principle of fluorescence resonance energy transfer (FRET). In this study, we develop a genetically programmable protein module that is easily adaptable for screening inhibitors for a wide range of proteases. The specific approach was to generate a quantum dot (QD)-modified, protease-specific protein module that can be used as a FRET substrate for probing protease activity. Intracellular delivery of the probes was facilitated by the use of a flanking TAT peptide and the site-specific incorporation of an acceptor fluorescent dye was accomplished using a unique cysteine residue. Presence of an elastin domain within the module enabled the simple purification of the QD-modified FRET substrate. For the initial testing, we developed a substrate peptide sequence that contains the cleavage site which is recognized by the polio viral protease PV2Apro. Utility of these new probes for monitoring viral activity and to screen for protease inhibitors will be discussed.

Background

Fluorescent Protein-Based FRET Pair

• Disadvantages of using organic fluorophores and fluorescent proteins include:

• narrow excitation bands• broad emission bands• low resistance to photo degradation

Methods

QD-Based Genetically Engineered Protein Module

Experimentation

Acknowledgements

We would like to thank the National Science Foundation, Dr. Victor Rogers, Denise Sanders, Jun Wang, andShen-Long Tsai

Protease

• Proteases are enzymes that catalyze the hydrolysis of peptide bonds, breaking down proteins

• Viral proteases are very important virulence factors in infection as they catalyze the hydrolysis of the longer polyprotein into functional enzymes for continuation of the viral lifecycle and infection

•Proteolysis is very specific and the viral proteases are highly expressed early on allowing for more rapid detection

FRET

• Fluorescence Resonance Energy Transfer

Nature Reviews Molecular Cell Biology 4; 579-586

Hwang et al., AEM. 72(5): 3710–3715 (2006)

Figure 2: Protein-based FRET pair. Protease cleavage results in the emission of CFP rather than YFP

Figure 1: Donor and acceptor FRET-pair

Quantum Dot-Based FRET Pair

• Advantages of using a quantum dot donor:• broad excitation bands• narrow emission bands• higher resistance to photo bleaching

• A disadvantage however lies in the inability for intracellular delivery of the conjugated protein into cells

Figure 3: Quantum Dot-Based FRET Pair before and after the protease’s cleavage of the linker sequence

Nature Materials 5, 581 - 589 (2006)

Elastin-Like Protein (ELP) Domain

• Repeating sequence {(VPGVG)2 (VPGKG) (VPGVG)2} 20

• Reversible temperature dependent precipitationELP

T>Tt T<Tt

NATURE METHODS | VOL.2 NO.9 | SEPTEMBER 2005 | 659.

Figure 4: Comparison of heated ELP-protein solution (left) with cool dissolved protein (right)

TAT Peptide

• Allows our QD protein to penetrate through cell membranes with minimal cell toxicity

• Cluster of basic amino acids mad up of 6 arginine and 2 lysine residues within a linear sequence of 9 amino acids (YGRKKRRQRRR)

Protein Expression

ELP precipitation and centrifuging

pure unconjugated protein module

48kD

Figure 5: Process of protein expression from cells containing the expression vector to the purified unconjugated protein

QD and Alexa Dye Conjugation

• Alexa 568 maleimide dye conjugation with protein module

• 2 hour incubation of protein module with thiol-reactive dye, Alexa 568 maleimide, followed by thermal ELP purification of conjugated protein modules

References

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QD emission Alexa568 absorption

CYS

Figure 6: Unconjugated Alexa 568 in the supernatant after three ELP purification cycles

Figure 7: Supernatant after three ELP cycles shows no unconjugated Alexa dye (left). Proteins conjugated to the Alexa dye pelleted down and suspended in 10mM Hepe’s Buffer (middle and right respectively)

Figure 8: Spectral overlap of QD emissions on Alexa 568 absorption

QD-Alexa Protein FRET Pair

Figure 10: Conjugated protein’s fluorescent emissions at different QD : Alexa ratios. Blue curve shows the emissions without conjugating the Alexa to the protein

QD : Alexa

Conjugated Protein Functionality

•Before Cleavage (blue): The QD is excited and its emissions are absorbed by the Alexa dye

•As the protein is cleaved (red) and FRET is disrupted, there is an increase in QD emissions and a decrease in Alexa emissions

After Cleavage

Before Cleavage

Hwang, Yu-Chen, Chen, Wilfred, Yates, Marylynn V. Use of Fluorescence Resonance Energy Transfer for Rapid Detection of Enteroviral Infection In VivoAppl. Environ. Microbiol. 2006 72: 3710-3715

Igor L. Medintz et al., Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot–peptide conjugates Nature Materials 5, 581 - 589 (2006)

Rüdiger Rudolf, Marco Mongillo, Rosario Rizzuto & Tullio Pozzan. Looking forward to seeing calcium Nature Reviews Molecular Cell Biology 4, 579-586 (July 2003)

Mahmoud Reza Banki, Liang Feng & David W Wood, Simple bioseparations using self-cleaving elastin-like polypeptide tags NATURE METHODS | VOL.2 NO.9 | SEPTEMBER 2005 | 659

Figure 11: Emissions after 3.5 hours of protease activity