GFP-V96 20 o CGFP-V96 40 o C GFP 40 o CGFP 20 o C Transition No Transition Development of a rapid, reversible protein switch in eukaryotes Studying how cells function requires methods that can both rapidly and reversibly arrest the target pathway in order to study the downstream effectors. With time, a cell can compensate for this loss of pathway, which may confound experimental results. We have therefore devised a new technique to address this problem using elastin-like polypeptides (ELPs), a type of environmentally responsive polypeptide derived from the human protein tropoelastin. ELPs are repeats of the amino acid sequence (Val- Pro-Gly-Xaa-Gly)n, where a guest residue, Xaa, can be virtually any amino acid and length, n, is variable. ELPs have a characteristic inverse phase transition temperature, Tt, above which they rapidly separate from aqueous solution. An ELP’s Tt depends on length, guest residue, and environment; any of which can be modulated to induce a phase transition. Consequently, the ELP can cycle between soluble and insoluble states, equivalent to an on/off switch. Our approach is to exploit this physical property of the ELP to reversibly shut down target cellular processes; however, first we have focused on characterizing the biophysics of ELPs inside live mammalian cells. M. Pastuszka, S. Janib, P. Hsueh, P. Shi, and J.A. Mackay Department of Pharmacology and Pharmaceutical Sciences, University of Southern California Background Results Conclusion Acknowledgments: USC School of Pharmacy Members of the Mackay lab NIH/NEI R01 EY S1 NIH/NCI CCSG 5 P30 CA Successfully designed, expressed and characterized fluorescent ELPs inside living cells Transitioned and imaged ELP polypeptides within mammalian cells Transition temperatures of GFP-ELPs are comparable to transition temperatures of free ELPs in PBS Soluble ELPs are mobile while aggregated ELPs are immobile in the cytoplasm Figure 1. ELP behavior before and after heating. A) Hypothesized clathrin shut-off B)Purified ELPs C)GFP labeled ELPs inside Hek293 cells. Hypothesis: GFP tagged ELP fusion proteins will transition rapidly and reversibly within the cytoplasm of a mammalian cell. To guide the design of ELP fusion switches, ELP libraries of varying length and Tt have been synthesized within bacterial cells. These ELPs have been fused to GFP and transfected into mammalian cell lines. The ELP phase transition has been observed using confocal scanning microscopy. Intracellular behavior will be characterized for: (i) ELP mobility before and after the phase transition using fluorescence recovery after photobleaching (FRAP) (ii) the dependence of transfection efficiency on ELP Tt (iii) the size and intracellular location of ELP aggregates. Methods 2 Fluorescence Recovery After Photobleaching (FRAP) demonstrates ELP mobility within the cytoplasm Figure 2. FRAP A. Example of FRAP. Cell expressing GFP-2VA were incubated at 40oC in order to induce aggregation of ELP. FRAP revealed that untransitioned GFP labeled ELP is more mobile than aggregated GFP-ELP. B. Plot of fluorescence intensity in a ROI versus time after photobleaching. The prebleach (Fpre) is calculated as the average of four images. F∞ is the asymptote of the recovery curve. Both Fpre and F∞ are used to calculate the mobile and immobile fractions. Transfection Efficiency is Guest Residue Dependent Figure 3. Transfection efficiency of Hek293 cells is ELP Tt dependent. The percentage of transfected cells is correlated to the transition temperature of 25μM free ELP in PBS. As ELP transition temperature increases due to increased fraction of hydrophilic residues the amount of transfected cells increases. GFP tagged ELPs transition with increased temperature Tt decreases as ELP length increases GFP-V60GFP-V72GFP-V96 40 o C 32 o C26 o C Figure 1. Demarcation of ELP cellular transition. (A) Free GFP retains its diffuse morphology with increased temperature. (B) ELP is diffuse throughout the cytoplasm of the cell at before transition. After transtioning, the GFP- ELP is observed as puncta throughout the cell, stronger intensity of GFP due to aggregation. Figure 2. GFP-ELP retains the same physical characteristics as free ELP in PBS. Increase in ELP length, results in a decrease in transition temperature. Amino Acid Sequence ELP MW (kD) GFP-ELP MW (kD) Tt of Free ELP in PBS ( o C) Tt of GFP- ELP in vitro ( o C) V60G(VPGVG) 60 Y V72G(VPGVG) 72 Y V96G(VPGVG) 96 Y A96G(VPGAG) 96 Y GFP-ELP Tt in vitro is comparable to ELP Tt in PBS