TEMPLATE DESIGN © A genetically programmable protein module as intracellularly deliverable QD-based FRET probes for viral protease detection Nikola Finneran, Divya Sivaraman, Payal Biswas, and Wilfred Chen Department of Chemical and Environmental Engineering, University of California, Riverside, CA, 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, and Shen-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; 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, (2006) Elastin-Like Protein (ELP) Domain Repeating sequence {(VPGVG) 2 (VPGKG) (VPGVG) 2 } 20 Reversible temperature dependent precipitation ELP T>T t T<T t 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 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 Vivo Appl. Environ. Microbiol : Igor L. Medintz et al., Proteolytic activity monitored by fluorescence resonance energy transfer through quantum- dot–peptide conjugates Nature Materials 5, (2006) Rüdiger Rudolf, Marco Mongillo, Rosario Rizzuto & Tullio Pozzan. Looking forward to seeing calcium Nature Reviews Molecular Cell Biology 4, (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