Tivadar Orban, Beata Jastrzebska, Sayan Gupta, Benlian Wang, Masaru Miyagi, Mark R. Chance, and Krzysztof Palczewski
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1 Structure, Volume Supplemental Information Conformational Dynamics of Activation for the Pentameric Complex of Dimeric G Protein-Coupled Receptor and Heterotrimeric G Protein Tivadar Orban, Beata Jastrzebska, Sayan Gupta, Benlian Wang, Masaru Miyagi, Mark R. Chance, and Krzysztof Palczewski Inventory of Supplemental Information Experimental methods : Purification of Rho Purification of G t Purification of the Rho* G t complex Table S. Sequences of peptic fragments from Rho s primary sequence showing normalized hydrogen deuterium uptake for Rho, Rho*, and Rho* G t complex, related to Figure Table S. Sequences of peptic fragments of G tα s primary sequence showing normalized, related to Figure Figure S. Hydrogen deuterium exchange exhibited by purified Rho and Rho* in the presence and absence of lipids, related to Figure 7 Figure S. Hydrogen deuterium exchange of Rho* in the presence of G t, related to Figure Figure S. Hydrogen deuterium exchange of G t α in the presence and absence of Rho*, related to Figure 9
2 Experimental methods Purification of Rho Following purification of Rho ZnCl was then removed by dialysis in the presence of. mm DDM. The purity of Rho, assessed spectrophotometrically from its absorption ratio at nm (protein peak) to nm (chromophore peak), was typically., suggesting that Rho was devoid of opsin and other proteins (Okada et al., ; Okada et al., 99). Rho also appeared homogenous after SDS PAGE analysis. The concentration of Rho was calculated by using the absorption coefficient =, M cm to quantify its absorption at nm (Matthews et al., 9). A typical yield was mg of Rho per bovine retinas. Lipid free Rho was purified by scona affinity resin prepared by coupling scona (Vector Laboratories, CA) to CNBr activated agarose (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at a density of mg scona/ml of resin. A column containing scona resin was equilibrated with mm BTP, ph.9, containing mm NaCl, mm MnCl, mm CaCl, mm MgCl, mm DTT and. mm DDM. ZnCl opsin extracted Rho was diluted to ~. mg/ml and loaded onto the column at. ml/min. The resin was washed with column volumes of equilibrating buffer and then Rho was eluted at. ml/min with the same buffer containing mm methyl D mannoside. The protein elution profile was examined by SDS PAGE and fractions containing Rho were pooled and concentrated with a, NMWL Centricon device to ~ mg/ml. Protein concentrations were determined as described above. Purification of G t Briefly, ROS membranes from retinas were diluted in ml of isotonic buffer ( mm HEPES, ph 7., mm MgCl, mm DTT and mm NaCl) and soluble proteins were
3 removed by gentle homogenization followed by centrifugation at,g at o C for min. G t then was extracted from the pellet in ml of hypotonic buffer ( mm HEPES, ph 7.,. mm EDTA and mm DTT), membranes were collected by centrifugation at,g at o C for min and the supernatant was saved for further purification. This extraction procedure was repeated three times. Combined supernatants were centrifuged at,g for min to remove ROS membrane contaminants, and then HEPES, ph 7., was introduced from a M stock solution to achieve a final concentration of mm and MgCl was added to a final concentration of mm. This solution was applied to ml of pentyl agarose resin equilibrated with buffer composed of mm HEPES, ph 7., mm MgCl and mm DTT at a flow rate of ml/h. The resin then was washed with column volumes of equilibrating buffer. Bound proteins were eluted with a ml linear gradient of to. M NaCl in the equilibrating buffer at a flow rate of ml/h, and ml fractions were collected. Fractions containing G t were pooled and concentrated by a, NMWL Centricon device (Millipore, Billerica, MA). G t was purified to homogeneity on a Superdex gel filtration column equilibrated with buffer containing mm HEPES, ph 7., mm NaCl, mm MgCl, and mm DTT at o C (flow rate. ml/min with collection of. ml fractions). Fractions containing G t were combined and concentrated by a, NMWL Centricon device to ~ mg protein/ml as determined by the Bradford assay (Bradford, 97). A typical purification yielded ~ mg of G t with 9% purity from bovine retinas. Purification of the Rho* G t complex Briefly, a column containing CNBr scona resin was equilibrated with buffer composed of mm BTP, ph.9, containing mm NaCl, mm MnCl, mm CaCl, mm MgCl, mm
4 DTT and. mm DDM. ZnCl opsin extracted Rho was diluted with this equilibrating buffer to ~. mg/ml and loaded on the column. The resin was washed with column volumes of the same buffer and then exposed for min to light located cm away ( Watt filter light, Dolan Jenner Industries Inc.) that passed through a nm band pass filter. Purified G t diluted to ~. mg/ml with equilibrating buffer was applied to the column immediately after light exposure. The resin was washed with column volumes of equilibrating buffer and bound protein was eluted with buffer composed of mm BTP, ph.9, containing mm NaCl, mm MnCl, mm CaCl, mm MgCl, mm DTT,. mm DDM and mm methyl D mannoside. The protein elution profile was examined by SDS PAGE and fractions containing the Rho* G t complex were pooled. This preparation was used directly for footprinting experiments or combined fractions were concentrated to ~ mg/ml with a, NMWL Centricon device (Millipore, Billerica, MA) for hydrogen deuterium exchange experiments. Concentrations of the purified Rho* G t complex were determined by Bradford ULTRA (Novexin, UK) with bovine serum albumin used as the standard.
5 Table S, related to Figure. Sequences of peptic fragments from Rho s primary sequence showing normalized hydrogen deuterium uptake for Rho, Rho*, and Rho* G t complex a. a Column shows the peptide sequence from the Rho primary sequence. Amino acid residues from the primary sequences of Rho found to have modifications such as attached carbohydrates, disulfide bonds, or palmitate are shown in bold underlined letters. Column exhibits the m/z of the ion used to identify the peptide based on the MS/MS spectrum, column indicates the charge of the ion from column, column shows the retention time (min) obtained from the HPLC chromatogram and used to identify the deuterated species, column features the number of theoretically exchangeable sites of the peptide fragment (see detailed calculation based on peptide sequence and sample dilution described in section Analysis of hydrogen deuterium exchange ), columns to indicate uptakes normalized to % of the theoretical maximum exchangeable sites (column ) of Rho with lipids, Rho in pure detergent, Rho* with lipids, Rho* in pure detergent, and of the Rho* Gt complex. Error values represent SDs obtained from three independent measurements. ND signifies unreliable detection of the peptide signal based on either overlap with other deuterated signals, an intensity lower than four times the preset mass spectrometer imposed baseline, or an absence from the spectrum. Sequence m/z z Retent ion time (min) Nbr. of excha ngeabl e sites (%) Rho lipids (% uptak e) Rho pure deterge nt (% uptake) Rho* lipids (% uptake ) Rho* pure detergent (% uptake) Rho* G t complex (% uptake) G TEGPNFYVP..-. ND ND ND ND 9.±. 9. V PFSNKTGVVRSPFE 9....±.±..± ND.±. APQYY Y LAEPWQFSMLAAY ±..±..±. 7.±. MFLLIMLGFPINF.7.9 L GFPINF 7....±.±..± 7..±...7. Y VTVQHKKLRTPLNY....7± ND.7±. 7.±..±. IL.. Y VTVQHKKLRTPLNY 9... ND.±.7 ND ND ND ILLNLAVADL. H KKLRTPLN.9.. ND ND ND ND 9.±. 7.7 A VADLF....9±.±..7± 7..±7.
6 7... L AVADLFMVFGGFTT.. 7. ND.±. ND 9.±. ND TLYTSLHG. F MVFGGFTTTL....±.±.9.±..±...7 T 9 TLYTSLHGYFVF.. 9. ND.±. ND.9±.7.±..7 C NLEGFFATLG 7....±.±..±..±..±. C 7 GIDY.. A 7 TLGGE 7....±.±..7±..±..7±..9. W SLVVL ±..±..±.7.9±.9.±. A IERYVVVCKPMSN ±..±..9±..±..±. F RFGENHAIMG ND.±..±7..±.7.±. R 7 FGENHAIMG.. F RFGEN 79.7 G VAFTWVMA 9. 7 A 9 CAAPPLVG 79. A 9 CAAPPLVGWSRYIP EGMQ ±.. ND.±7...± 7.±. 7...±.±.. 9 ND.±. 9 ND ND ND ND ND ND ND ND.±7. ±..9±.±... ND 9..7 W 7 SRYIPEGMQ ±..±. 7.±..±.9.±. Y 9 YTPHEETNNESF ±.9 7.9±. 7.±.9.±..7±. FIIPLI..-. ND ND ND.±..±.. F CYGQLVF ±.±. 7.±.±. ND
7 .9.. A TTQKAEK ±.±..±..7±. ND Q 7 QESATTQKAEKEV... ND 9.7±. ND 9.±.9.9±.7 TRMVIIMVIAFLIC 9. A FLICWLPYA ±..±.. ±.. Y 7 IFTHQGSDFGPIF. 77. M TIPAF 79. F 9 AKTSAVYNPVIY 7. 9 N KQFRNCMVTTLCC. GKNPLGDDE T VSKTETSQVAPA. V 7 SKTETSQVAPA ±...±.. 9.7±...±.9. 7.±.. 7.±.7 7.±. 7.±..±..± ±. 7.7±. 9.±..±.7.±..±..±..±.9.9±9 7.±..9±...±.7±.9.±...±.±.9.±.. ND ND.7±. 7
8 Table S, related to Figure. Sequences of peptic fragments of G tα s primary sequence showing normalized hydrogen deuterium uptake for G tα free or in complex with Rho*. Column indicates the peptide sequence from the primary sequence of G tα. Column shows the m/z of the ion used to identify the peptide based on the MS/MS spectrum, column exhibits the charge of the ion from column, column indicates the retention time (min) obtained from the HPLC chromatogram and used to identify the deuterated species, column reveals the number of theoretically exchangeable sites of the peptide fragment (see detailed calculation based on peptide sequence and sample dilution described in section Analysis of hydrogen deuterium exchange ), column denotes uptake normalized to % of the theoretical maximum exchangeable sites (column ) of G tα whereas column 7 shows normalized uptake of G tα in the presence of Rho*. Error values represent SDs obtained from three independent measurements. ND signifies unreliable detection of the peptide signal based on either overlap with other deuterated signals, an intensity lower than four times the preset mass spectrometer imposed baseline, or an absence from the spectrum. Sequence m/z z RT (min) Nr. of exchan geable sites (%) A 7 EEKHSRELEKKLKEDAEKDART 9. L LLGAGESGKSTIVKQ. L LLGAGESGKSTIVKQMKIIHQDG. YSLE I 7 AIIYGNTLQ. 7 A IVRAMTTL 97.. N IQYGDSARQDDARKLMHMAD.7 W 7 KDSGIQAC 7. 7 F 7 DRASE 7. Gt (% uptake) Rho*- Gt (%upta ke) ±..±..-...±7..± ±..± ±..±..-...±. ND...7± ±..7±..-...±..±. Y QLNDSAG ±.7 9.±.
9 E 7 RLVTPGYVPTEQD ±..±9. V 7 LRSRVKTTGIIE ±. 7.±. S 7 FKDLNF ±..±. K WIHCFEGVTCIIFIAALSAY ±..±. V EDDEVNRMHESLHL ±..±. 9. F 7 NSICNHRYFATTS..-...±.9 7.±. F LNKKDVFSEKIKKAHL..-...±. 9.±. Y 99 IKVQF N MRRDVKEIYSHMTC 9. T CATDTQNVKFVF 77.7 A TDTQNVKFVF V FDAVTD 7. D IIIKENLKDCGL ±..± ±..± ±. 7.± ND.±...7±.7.± ±. 9.±. 9
10 Figure S, related to Figure 7. Hydrogen deuterium exchange exhibited by purified Rho and Rho* in the presence and absence of lipids. Panels A and B show deuterium uptake of purified Rho and Rho* in the presence of lipids. Panels C and D illustrate deuterium uptake of Rho and Rho* in the absence of lipids. A two-dimensional plot of the Rho primary sequence is used to represent the normalized uptake identified for the peptic fragments. After representation of the actual percent uptake as a function of D O incubation time (typical plots are shown in Fig. A F), values were corrected to account for sample dilution (see section Hydrogen deuterium exchange analysis for a detailed description in Materials and Methods). Normalized values of the uptake obtained after min incubation in D O are recorded in Table S (columns to ) and these were used for color coding of Rho s primary sequence. Color coding is shown as 9%; blue, 9%; light blue, 9%; cyan, 9%; green, 9%; yellow, 9%; orange, 9%; red, 7 79%; pink, -%
11 Figure S, related to Figure. Hydrogen deuterium exchange of Rho* in the presence of G t. This figure shows the deuterium uptake of Rho peptic peptide fragments obtained from Rho* G t samples. Calculation of the normalized deuterium uptake and the color-coding employed are described in Fig. S.
12 Figure S, related to Figure 9. Hydrogen deuterium exchange of G t α in the presence and absence of Rho*. Panel A shows the recent model of Rho*-G t () with the mapped hydrogen deuterium exchange profile of the α domain of G t in the presence of Rho*. Calculation of the normalized deuterium uptake is based on Table S whereas the color-coding used is described in Fig. S. The Rho* dimer together with G tβ and G tγ domains are shown as gray cartoons. Panel B illustrates a 9 rotated view of the G tα domain shown in Panel A. The G t AH and G t Ras subdomains are labeled appropriately. Panel C shows the deuterium uptake profile of G t α in the absence of Rho*.
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