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Using synthetic lipids to stabilize purified β2 adrenoceptor in detergent micelles
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 343 (2005) 344–346 www.elsevier.com/locate/yabio
Notes & Tips
Using synthetic lipids to stabilize puriWed 2 adrenoceptor in detergent micelles Zhiping Yao, Brian Kobilka ¤ Stanford University School of Medicine, Stanford, CA 94305, USA Received 1 March 2005 Available online 25 May 2005
G protein-coupled receptors (GPCRs)1 mediate the majority of cellular responses to hormones and neurotransmitters. Consequently, they constitute the largest family of pharmaceutical targets. EVorts to apply structure-based drug design and in silico screening to facilitate drug discovery for this important class of molecules is limited by the lack of high-resolution structural information. Bovine rhodopsin is the only GPCR for which a high-resolution structure is available [1]. This remarkable achievement was accomplished using a natural source of protein and an elegantly simple puriWcation procedure [2]. The natural abundance of rhodopsin was clearly a factor in the successful crystallography eVorts, but also important is the remarkable structural stability of this protein relative to other GPCRs. This stability is attributable in part to the stabilizing eVect of the bound ligand 11-cis-retinal. Another factor that has been proposed to contribute to successful crystallization is the fact that the process of puriWcation was limited to a simple detergent extraction [2]. Therefore, the puriWed protein likely contained a signiWcant amount of natural lipids as mixed lipid–detergent micelles. The 2 adrenergic receptor (2AR) is one of the most extensively characterized GPCRs. Moreover, methods have been developed for the production and puriWcation of this receptor in milligram quantities [3]. Therefore, it
*
Corresponding author. Fax: +1 650 498 5092. E-mail address: [email protected] (B. Kobilka). 1 Abbreviations used: GPCR, G protein-coupled receptor; 2AR, 2 adrenergic receptor; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPS, 1,2dioleoyl-sn-glycero-3-[phospho-L-serine]; UPC, unsaturated phosphatidylcholine; HLS, Hepes low-salt buVer; DHA, docosahexaenoic acid; CHS, cholesterolhemisuccinate; PC, phosphatidylcholine. 0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.05.002
is a candidate for crystallography eVorts; however, the stability of the 2AR in detergent solution at room temperature is limited. During the three chromatography steps involved in 2AR puriWcation, it is likely that most, if not all, of the natural lipids are stripped from the receptor protein. Therefore, we examined the eVect of synthetic lipids on the stability of puriWed 2AR. Here we report that the addition of relatively small amounts of speciWc lipids to dodecylmaltoside micelles can signiWcantly prolong the lifetime of the 2AR in solution. Cholesterol hemisuccinate was purchased from Steraloids (Newport, RI, USA). Other lipids, including polar liver extract, polar brain extract, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), unsaturated phosphatidylcholine (UPC), and 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, were purchased from Avanti Polar Lipids (Alabaster, AL, USA) as chloroform solutions. The desired amount of lipid solution was placed in a glass vial, and chloroform was evaporated oV under a constant stream of argon. To prepare the lipid solution in detergent-containing buVer, the dried lipid was suspended in Hepes low-salt buVer (HLS) consisting of 20 mM Hepes (pH 7.5), 100 mM NaCl, and 0.1% dodecylmaltoside (Anatrace, Maumee, OH, USA), by sonication for 15 min at 20 °C. For each lipid, a stock solution was prepared having a lipid:detergent ratio of 1:1 (w/w). Additional lipid:detergent mixtures were prepared by dilution with HLS. 2AR was puriWed by a three-step chromatography procedure involving FLAG antibody aYnity chromatography, alprenolol ligand aYnity chromatography, and nickel chromatography, as described previously [4]. This procedure yields puriWed 2AR at a concentration
Notes & Tips / Anal. Biochem. 343 (2005) 344–346
of 5–10 m in a buVer consisting of HLS with 200 mM imidazole. The concentrated receptor was diluted 250fold into the lipid–detergent mixtures or into HLS as a control. Initial 2AR activity was determined by [3H]docosahexaenoic acid (DHA) binding assay, as described previously [3]. Samples were then incubated
Fig. 1. EVects of various synthetic lipids on the stability of the 2AR. PuriWed 2AR was diluted 250-fold into HLS containing lipid at the indicated detergent:lipid ratio. The white bars indicate binding activity determined immediately after dilution, and the black bars indicate binding activity after incubation at 37 °C for 4 h. Data represent the means and standard deviations of experiments performed in triplicate.
345
for varying times and at varying temperatures, and 2AR activity was determined by binding to a saturating (10 nM) concentration of [3H]DHA. For each condition, Wve total and three nonspeciWc binding determinations were made. Each experiment was repeated three times. The 2AR was expressed in SF9 insect cells and puriWed by three successive chromatography steps as described above. Fig. 1 shows the eVect of Wve diVerent lipids on 2AR stability at 37 °C (black bars). All lipids signiWcantly enhanced receptor stability. The greatest eVects were observed at detergent:lipid ratios of 1.0:0.3. Of interest, we observed a trend of higher 2AR binding activity in receptor diluted into detergent-containing lipids even before incubation at 37 °C (white bars). This diVerence was signiWcant for the 1.0:0.3 mixture of detergent:DOPS (P < 0.001). This eVect may be due to lipidmediated protection of the 2AR against the denaturing eVects of diluting the receptor 250-fold for the binding
Fig. 2. (A) Comparison of the eVect of various lipids on the stability of 2AR to incubation at 37 °C for 4 h. All lipids were used at a detergent:lipid ratio of 1.0:0.3. Binding activity is relative to 0 h control of receptor in detergent alone. Values for all lipids were signiWcantly diVerent from the value for the detergent control (P < 0.05). (B) EVects of CHS on 2AR stability at room temperature and 4 °C. Receptor was incubated in HLS buVer or HLS buVer with CHS at a detergent:lipid ratio of 1.0:0.3 at either room temperature (20–25 °C) or 4 °C for 2 and 7 days. Binding activity is relative to 0 h control of receptor in detergent alone. Data represent the means and standard deviations of experiments performed in triplicate.
346
Notes & Tips / Anal. Biochem. 343 (2005) 344–346
assay. Alternatively, DOPS may be able to refold some incompletely denatured receptor. We performed an experiment in which all lipids were compared at the same time using the same preparation of protein (Fig. 2A). Although all lipids provided signiWcant protection, the greatest eVect on receptor stability was observed with cholesterolhemisuccinate (CHS), and the smallest eVect was observed with DOPE. We also examined the eVect of polar lipid extracts from liver and brain; these were not found to provide signiWcantly more protection than CHS (Suppl. Fig. 1). The eVect of CHS was not enhanced by the inclusion of phosphatidylcholine (PC) or UPC (data not shown). Based on these results, we examined long-term eVects of CHS on 2AR stability at room temperature (20–25 °C) and 4 °C for 2 and 7days (Fig. 2B). SigniWcant stabilizing eVects were observed at both temperatures. In conclusion, we examined the ability of synthetic lipids (DOPC, DOPE, DOPS, UPC, and CHS) to stabilize detergent-solubilized 2AR. The greatest eVect on receptor stability was observed with CHS and UPC, whereas DOPE had the smallest stabilizing eVect. The optimal ratio of detergent to lipid was found to be 1.0:0.3
(w/w) for all lipids. These results are likely to apply to other GPCRs as well. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.ab.2005.05.002. References [1] K. Palczewski, T. Kumasaka, T. Hori, C.A. Behnke, H. Motoshima, B.A. Fox, I. Le Trong, D.C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto, M. Miyano, Crystal structure of rhodopsin: a G protein-coupled receptor, Science 289 (2000) 739–745. [2] T. Okada, I. Le Trong, B.A. Fox, C.A. Behnke, R.E. Stenkamp, K. Palczewski, X-ray diVraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles, J. Struct. Biol. 130 (2000) 73–80. [3] B.K. Kobilka, Amino and carboxyl terminal modiWcations to facilitate the production and puriWcation of a G protein-coupled receptor, Anal. Biochem. 231 (1995) 269–271. [4] S. Devanathan, Z. Yao, Z. Salamon, B. Kobilka, G. Tollin, Plasmon-waveguide resonance studies of ligand binding to the human beta 2-adrenergic receptor, Biochemistry 43 (2004) 3280–3288.
Notes & Tips
Using synthetic lipids to stabilize puriWed 2 adrenoceptor in detergent micelles Zhiping Yao, Brian Kobilka ¤ Stanford University School of Medicine, Stanford, CA 94305, USA Received 1 March 2005 Available online 25 May 2005
G protein-coupled receptors (GPCRs)1 mediate the majority of cellular responses to hormones and neurotransmitters. Consequently, they constitute the largest family of pharmaceutical targets. EVorts to apply structure-based drug design and in silico screening to facilitate drug discovery for this important class of molecules is limited by the lack of high-resolution structural information. Bovine rhodopsin is the only GPCR for which a high-resolution structure is available [1]. This remarkable achievement was accomplished using a natural source of protein and an elegantly simple puriWcation procedure [2]. The natural abundance of rhodopsin was clearly a factor in the successful crystallography eVorts, but also important is the remarkable structural stability of this protein relative to other GPCRs. This stability is attributable in part to the stabilizing eVect of the bound ligand 11-cis-retinal. Another factor that has been proposed to contribute to successful crystallization is the fact that the process of puriWcation was limited to a simple detergent extraction [2]. Therefore, the puriWed protein likely contained a signiWcant amount of natural lipids as mixed lipid–detergent micelles. The 2 adrenergic receptor (2AR) is one of the most extensively characterized GPCRs. Moreover, methods have been developed for the production and puriWcation of this receptor in milligram quantities [3]. Therefore, it
*
Corresponding author. Fax: +1 650 498 5092. E-mail address: [email protected] (B. Kobilka). 1 Abbreviations used: GPCR, G protein-coupled receptor; 2AR, 2 adrenergic receptor; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPS, 1,2dioleoyl-sn-glycero-3-[phospho-L-serine]; UPC, unsaturated phosphatidylcholine; HLS, Hepes low-salt buVer; DHA, docosahexaenoic acid; CHS, cholesterolhemisuccinate; PC, phosphatidylcholine. 0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.05.002
is a candidate for crystallography eVorts; however, the stability of the 2AR in detergent solution at room temperature is limited. During the three chromatography steps involved in 2AR puriWcation, it is likely that most, if not all, of the natural lipids are stripped from the receptor protein. Therefore, we examined the eVect of synthetic lipids on the stability of puriWed 2AR. Here we report that the addition of relatively small amounts of speciWc lipids to dodecylmaltoside micelles can signiWcantly prolong the lifetime of the 2AR in solution. Cholesterol hemisuccinate was purchased from Steraloids (Newport, RI, USA). Other lipids, including polar liver extract, polar brain extract, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), unsaturated phosphatidylcholine (UPC), and 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, were purchased from Avanti Polar Lipids (Alabaster, AL, USA) as chloroform solutions. The desired amount of lipid solution was placed in a glass vial, and chloroform was evaporated oV under a constant stream of argon. To prepare the lipid solution in detergent-containing buVer, the dried lipid was suspended in Hepes low-salt buVer (HLS) consisting of 20 mM Hepes (pH 7.5), 100 mM NaCl, and 0.1% dodecylmaltoside (Anatrace, Maumee, OH, USA), by sonication for 15 min at 20 °C. For each lipid, a stock solution was prepared having a lipid:detergent ratio of 1:1 (w/w). Additional lipid:detergent mixtures were prepared by dilution with HLS. 2AR was puriWed by a three-step chromatography procedure involving FLAG antibody aYnity chromatography, alprenolol ligand aYnity chromatography, and nickel chromatography, as described previously [4]. This procedure yields puriWed 2AR at a concentration
Notes & Tips / Anal. Biochem. 343 (2005) 344–346
of 5–10 m in a buVer consisting of HLS with 200 mM imidazole. The concentrated receptor was diluted 250fold into the lipid–detergent mixtures or into HLS as a control. Initial 2AR activity was determined by [3H]docosahexaenoic acid (DHA) binding assay, as described previously [3]. Samples were then incubated
Fig. 1. EVects of various synthetic lipids on the stability of the 2AR. PuriWed 2AR was diluted 250-fold into HLS containing lipid at the indicated detergent:lipid ratio. The white bars indicate binding activity determined immediately after dilution, and the black bars indicate binding activity after incubation at 37 °C for 4 h. Data represent the means and standard deviations of experiments performed in triplicate.
345
for varying times and at varying temperatures, and 2AR activity was determined by binding to a saturating (10 nM) concentration of [3H]DHA. For each condition, Wve total and three nonspeciWc binding determinations were made. Each experiment was repeated three times. The 2AR was expressed in SF9 insect cells and puriWed by three successive chromatography steps as described above. Fig. 1 shows the eVect of Wve diVerent lipids on 2AR stability at 37 °C (black bars). All lipids signiWcantly enhanced receptor stability. The greatest eVects were observed at detergent:lipid ratios of 1.0:0.3. Of interest, we observed a trend of higher 2AR binding activity in receptor diluted into detergent-containing lipids even before incubation at 37 °C (white bars). This diVerence was signiWcant for the 1.0:0.3 mixture of detergent:DOPS (P < 0.001). This eVect may be due to lipidmediated protection of the 2AR against the denaturing eVects of diluting the receptor 250-fold for the binding
Fig. 2. (A) Comparison of the eVect of various lipids on the stability of 2AR to incubation at 37 °C for 4 h. All lipids were used at a detergent:lipid ratio of 1.0:0.3. Binding activity is relative to 0 h control of receptor in detergent alone. Values for all lipids were signiWcantly diVerent from the value for the detergent control (P < 0.05). (B) EVects of CHS on 2AR stability at room temperature and 4 °C. Receptor was incubated in HLS buVer or HLS buVer with CHS at a detergent:lipid ratio of 1.0:0.3 at either room temperature (20–25 °C) or 4 °C for 2 and 7 days. Binding activity is relative to 0 h control of receptor in detergent alone. Data represent the means and standard deviations of experiments performed in triplicate.
346
Notes & Tips / Anal. Biochem. 343 (2005) 344–346
assay. Alternatively, DOPS may be able to refold some incompletely denatured receptor. We performed an experiment in which all lipids were compared at the same time using the same preparation of protein (Fig. 2A). Although all lipids provided signiWcant protection, the greatest eVect on receptor stability was observed with cholesterolhemisuccinate (CHS), and the smallest eVect was observed with DOPE. We also examined the eVect of polar lipid extracts from liver and brain; these were not found to provide signiWcantly more protection than CHS (Suppl. Fig. 1). The eVect of CHS was not enhanced by the inclusion of phosphatidylcholine (PC) or UPC (data not shown). Based on these results, we examined long-term eVects of CHS on 2AR stability at room temperature (20–25 °C) and 4 °C for 2 and 7days (Fig. 2B). SigniWcant stabilizing eVects were observed at both temperatures. In conclusion, we examined the ability of synthetic lipids (DOPC, DOPE, DOPS, UPC, and CHS) to stabilize detergent-solubilized 2AR. The greatest eVect on receptor stability was observed with CHS and UPC, whereas DOPE had the smallest stabilizing eVect. The optimal ratio of detergent to lipid was found to be 1.0:0.3
(w/w) for all lipids. These results are likely to apply to other GPCRs as well. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.ab.2005.05.002. References [1] K. Palczewski, T. Kumasaka, T. Hori, C.A. Behnke, H. Motoshima, B.A. Fox, I. Le Trong, D.C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto, M. Miyano, Crystal structure of rhodopsin: a G protein-coupled receptor, Science 289 (2000) 739–745. [2] T. Okada, I. Le Trong, B.A. Fox, C.A. Behnke, R.E. Stenkamp, K. Palczewski, X-ray diVraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles, J. Struct. Biol. 130 (2000) 73–80. [3] B.K. Kobilka, Amino and carboxyl terminal modiWcations to facilitate the production and puriWcation of a G protein-coupled receptor, Anal. Biochem. 231 (1995) 269–271. [4] S. Devanathan, Z. Yao, Z. Salamon, B. Kobilka, G. Tollin, Plasmon-waveguide resonance studies of ligand binding to the human beta 2-adrenergic receptor, Biochemistry 43 (2004) 3280–3288.