Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio Mathew Tantama, Juan Ramón Martínez-François, Rebecca Mongeon, Gary Yellen* Department of Neurobiology, Harvard Medical School, Longwood Avenue, Boston, Massachusetts, 115; USA *gary_yellen@hms.harvard.edu; (ph) 17-3-137; (fax) 17-3-139 Supplementary Information
Supplementary Figure S1. Amino acid sequence alignment highlighting mutations. Protomers A-C refer to the repeats of the GlnK monomer that are attached in tandem in the Perceval sensors. Perc, original Perceval (K R.5, F max /F ). Interm, an intermediate mutant that showed an increased dynamic range but no change in K R (K R.5, F max /F > 8). PercHR, the final PercevalHR (K R 3.5, F max /F > 8). cpmv, cp15 circularly permuted Venus. The Linker between protomers is indicated. Libraries were screened with positive selection for improvements in K R and F max /F ; however, extensive sequencing and intermediate characterization was not performed to specifically evaluate the correlation between sequence changes and variation in sensor characteristics. Point mutations V3F (Protomer A) and V1P (Protomer C) are likely major determinants of the increased fluorescence response and shifted K R, respectively; however, we have not determined the precise contribution of each mutation to the improved characteristics of PercevalHR relative to the original Perceval. Protomer A: 1 3 3 5 11 ----*-----------------------------*------*------*------------------------------------------------------------------- GlnK mkkveaiirpekleivkkalsdagyvgmtvsevkgrgvqggiveryrgrey------ivdlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipvervvrvrtkeegkeal Perc mkkvesiirpekleivkkalsdagyvgmtvsevkgtgvqggiveryrgrey-cpmv-ivdlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipvervvrvrtkeegasgggsggggasg Interm mkkvesiirpekleivkkalsdagyvgmtvsevkgsgvqggiferyrgrvy-cpmv-ivdlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipvervvrvrtkeegasgggsggggasg PercHR mkkvesiirpekleivkkalsdagyvgmtvsevkgsgvqggiferyrgrvy-cpmv-ivdlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipvervvrvrtkeegasgggsggggasg Linker Protomer B: 38 39 99 -------------------------------------**-----------------------------------------------------------------*------------- GlnK mkkveaiirpekleivkkalsdagyvgmtvsevkgrgvqggiveryrgrey------ivdlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipvervvrvrtkeegkeal Perc mkkveaiirpekleivkkalsdagyvgmtvsevkgrgaggg------------------dlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipvervvrvrtkeegasgggggsggasg Interm mkkveaiirpekleivkkalsdagyvgmtvsevkgrgaggg------------------dlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipvervvrvrtkeegasgggggsggasg PercHR mkkveaiirpekleivkkalsdagyvgmtvsevkgrgaggg------------------dlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipverivrvrtkeegasgggggsggasg Linker Protomer C: 1 3 38 39 73 1 --------------------*-*--------------**---------------------------------------*----------------------------*---------- GlnK mkkveaiirpekleivkkalsdagyvgmtvsevkgrgvqggiveryrgrey------ivdlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipvervvrvrtkeegkeal Perc mkkveaiirpekleivkkalsdagyvgmtvsevkgrgaggg------------------dlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipvervvrvrtkeegkeal Interm mkkveaiirpekleivkkalsdagyvgmtvsevkgrgaggg------------------dlipkvkielvvkeedvdnvidiicenartgnpgdgkifvipvervvrvrtkeegkeal PercHR mkkveaiirpekleivkkalnddgyvgmtvsevkgrgaggg------------------dlipkvkielvvkeedvdniidiicenartgnpgdgkifvipvervvrprtkeegkeal
A B C 1. ATP Binding T-Loop Closure.8.... Control 37 C 1 mm KG 1 mm GTP 1 mm AMP ATP:ADP ratio D F high /F low EDTA Mg +.1 1 1 1 1 [ATP] (µm) E Adjusted F high /F low.5 mm Mg +.5 mm Mg + 1.5 mm Mg +..5 1. MgATP Occupancy F F low /F high 3 1.1 EDTA.5 mm Mg +.5 mm Mg + 1.5 mm Mg +.1 1 1 1 [free ADP] (µm) 1 Supplementary Figure S. PercevalHR sensor properties and basic cellular response properties. (A) Crystal structure of GlnK trimer. PDB J9C. (B) View of a single monomer. T-Loop closure induced by ATP binding. Blue: PDB J9C; no ATP. Red: PDB J9D; ATP, orange sticks; Mg +, yellow dotted sphere. (C)-(F) Characterization of purified sensor protein. (C) ATP:ADP doseresponse in the presence of 1 mm -ketoglutarate, 1 mm AMP, or 1 mm GTP. (D-F) Mg + - dependent binding of ATP. For simplicity we refer to "ATP:ADP" as the sensor's analyte; however, strictly speaking, the ATP:ADP ratio is a function of [Mg + ], [nucleotide] free, [MgNucleotide], and the of PercevalHR is a function of [MgATP], [ATP] free, and [ADP] free. Thus, changes in [Mg ] can alter nucleotide equilibria and be reflected in the sensor. (D) Dose responses in the presence of saturating 1.5 mm MgCl (red) or 1 mm EDTA (black). (E) PercevalHR response is formally a function of by MgATP, ATP, and ADP. The dose-response fits well to a four state model in which apo-percevalhr is in equilibrium with ATP-PercevalHR, ADP-PercevalHR, and MgATP-PercevalHR, where MgATP- PercevalHR exhibits a maximal F high /F low ratio greater than the minimum F high /F low ratio measured for ATP-PercevalHR and ADP-PercevalHR states. The "Adjusted F high /F low " takes into account the decrease in signal observed when free ATP binds Perceval seen in (D). (F) Binding of free ADP. Mg + can chelate ADP, reducing the effective free concentration. [free ADP] was calculated from [total ADP] and [Mg + ] using WebmaxC (http://www.stanford.edu/~cpatton/webmaxce.htm).
normalized Original Perceval normalized PercevalHR A 5 3 1 1.5.1 1mM glucose Supplementary Figure S3. Improved performance of PercevalHR compared to the original Perceval when expressed in live cells. (A) The original Perceval sensor or (B) the optimized PercevalHR sensor was expressed in NeuroA neuroblastoma cells and imaged at 31 3 C. Extracellular [glucose] was varied. PercevalHR detected changes in ATP:ADP coupled to changes in [glucose], and also detected two populations with different K apparent for the ATP:ADP versus [glucose] relationship (blue, N=; green, N=3); however, the original Perceval was not sensitive enough to detect these physiological changes (black, N=5). Sensor signals were ph-corrected and normalized to the ADP-saturated values (obtained with metabolic inhibition at the end of the experiment) in order to illustrate the difference in fluorescence dynamic range. Traces are means and error bars are standard deviations. B 5 3 3 9 1 1.5.1 1mM glucose 3 9
ph-corrected Signal A 1..5.5.5 1 8 mm glucose neuron. astrocytes C F high /F low typical range in experiment ATP:ADP B 15 9 7 8 ph ph calibration K R 1 5 3 DF max /F initial 1. ATP:ADP 7 8 ph.5. 7 8 ph Supplementary Figure S. (A) ATP:ADP did not change appreciably with changing [glucose] when astrocytes were not pre-incubated in low glucose imaging solution (N=17). This phenotype may be due to astrocytic glycogen stores. (B) ph dependence of the K R and the fluorescence dynamic range. (C) Example of the raw PercevalHR signal versus ph at different sensor occupancies (top) and the corrected Perceval HR signal versus ph after approximate ph correction (bottom). The ph calibration step removes a significant component of the ph bias and works relatively well for the ph range typically observed in experiments. In principle, using an absolute ph calibration, a second order ph correction could be performed to completely remove ph bias; however, we found that the approximate correction works very well because ph changes are mild on average. Red, ATP:ADP = ; green, ATP:ADP = ; blue ATP:ADP =.
1..15.15 ATP:ADP. 1 1.5.1.1. 1...15..15.5.1.1. 1...15..15.5.1.1.. 15 3...5 1. PercevalHR Supplementary Figure S5. Simultaneous PercevalHR imaging and cell-attached, single-channel patch-clamp recording from intact HEK93 cells expressing K ATP channels that were metabolically inhibited with 1 mm DG. The top panels and right panels are the same data shown in main Figure 5. The left bottom two panels represent timecourse data for the second and third cells whose data are shown in main Figure 5(C). Arrows indicated application of nm glibenclamide. Left panels show ph-corrected PercevalHR (green; left axis) and K ATP single-channel P open (black; right axis) as a function of time for individual cells. Right panels show K ATP single-channel P open (left axis) versus ph-corrected PercevalHR (bottom axis) for the associated left panel; the estimated ATP:ADP is also shown (top axis).
1..15.15 ATP:ADP. 1 1.5.1.1. 1...15..15.5.1.1. 1...15..15.5.1.1. 7 1....5 1. PercevalHR Supplementary Figure S. Simultaneous PercevalHR imaging and cell-attached, single-channel patch-clamp recording from intact HEK93 cells expressing K ATP channels that were metabolically inhibited with 1 mm IAA. The top panels and right panels are the same data shown in main Figure. The left bottom two panels represent timecourse data for the second and third cells whose data are shown in main Figure (C). Arrows indicated application of nm glibenclamide. IAA application caused cell health to deteriorate quickly, and in two of the three experiments the patch was lost before glibenclamide application. Left panels show phcorrected PercevalHR (green; left axis) and K ATP single-channel P open (black; right axis) as a function of time for individual cells. Right panels show K ATP single-channel P open (left axis) versus ph-corrected PercevalHR (bottom axis) for the associated left panel; the estimated ATP:ADP is also shown (top axis).
Supplementary Table S1. PercevalHR extinction coefficients and quantum yields determined as previously described 7. Values are mean standard deviation for n=3. Quantum yields were measured relative to a fluorescein reference standard in.1 M NaOH ( Fluorescein =.95 59 ; refractive index n = 1.33) and cross-verified using a rhodamine 13 reference standard in ethanol ( rhodamine13 =.9 ; n = 1.3). Extinction Coefficient (M -1 cm -1 ) Quantum Yield (unitless) 15nm 5nm 15nm 5nm ADP-Loaded 3 1 1..3.3.3 MgATP-Loaded 17 1 3.19.1.15.3 Supplementary References: 59. Brannon, J.H., Magde, D. Absolute quantum yield determination by thermal blooming. Fluorescein. J. Phys. Chem.8, 75-79 (1978).. Kubin, R.F., Fletcher, A.N. Fluorescence quantum yields of some rhodamine dyes. J. Luminescence 7, 55- (198).