Lot showed inclusion body (IB) and membrane fractions (M) of OPRM.

Lot showed inclusion body (IB) and membrane fractions (M) of OPRM. doi:10.1371/journal.pone.0056500.gConfirmation of Full Length of OPRMOPRM, western blot positive for the N-terminal his-tag, was found at a position of around 38 kDa on 12 SDS-PAGE (Figure 4), though the expected Mw is 46 kDa. Several integral membrane proteins including several GPCRs were found to migrate anomalously smaller than expected on SDS AGE due toOPRM from E. coliFigure 2. Growth conditions of OPRM in different E.coli strains. Expression of OPRM was induced by IPTG. Cell culture density (OD600) and weight of cell pellet (g) after different induction times with two different media (TB and DYT) was measured. Cell pellet (g) was obtained from 1 liter of culture medium. doi:10.1371/journal.pone.0056500.gtheir hydrophobicity and compact structure [30]. Nevertheless, the presence of the full-length protein had to be confirmed. The protein was extracted from SDS-PAGE, digested with trypsin and treated with iodoacetamide and DTT for analysis by mass spectrometry. Only after treatment with DTT and iodoacetamide before digestion with trypsin peptide matches were found (Figure 6A). Four matches were further analyzed by MS/ MS analysis. These peptides were derived from cytoplasmic and intracellular loops connecting transmembrane domains, but not from the N-terminal domain that does not contain a trypsin cleavage site. A total of 13 sequence PLV-2 coverage was obtained (Figure 6B). As the C-terminal peptide was also found, the band with apparent Mw of 38 kDa in SDS-PAGE corresponded to the full length of the 46 kDa protein.isolated in Peak 1 (Figure 5) was found to have an alpha-helical content corresponding to 5? TM-helices (data not shown).Confirmation of Receptor Function by Agonist BindingThe functionality of the isolated OPRM was probed by measuring the binding of the natural HIF-2��-IN-1 site ligand endomorphine-1 to OPRM by plasmon surface resonance. Initially about 8000 RUs of OPRM (MW 46 kDa) were bound to the Ni-NTA chip. After extensively washing with buffer ca. 4000 RU remained. These results illustrated that for membrane proteins high initial responses may be SC-66 web observed because of unspecific binding or MedChemExpress Hesperidin aggregation. The addition of reducing agent (1 mM TCEP) to the loading buffer did not change the binding of OPRM. Upon supplying increasing concentrations of agonist EM-1 to the immobilized OPRM increasing binding signal (RU) was observed (Rmax = 40 RU (EM-1: MW 610 Da)). Evaluation with a 1:1 interaction model allowed determining a KD of 61618 nM for the binding of EM-1 to OPRM isolated in detergent FOS-12 (Figure 8), which confirmed the agonist binding capacity of the isolated OPRM. No binding of endomorphine-1 was observed for reduced OPRM, which was immobilized on the chip in 1 mM TCEP. This negative control indicated that the endomorphine-1 binding pocket was stabilized by a disulfide-bond in OPRM.Confirmation of 7-TM Alpha-helical Secondary StructureA first 12926553 characterization of OPRM receptor natively purified from bacterial membrane was carried out by circular dichroism. The secondary structure of the purified OPRM after gel filtration was determined by CD-data from the far-UV spectrum in the 200?50 nm range (Figure 7) by K2D deconvolution. The folded protein was characterized to have a secondary structure of 4665 alpha-helix. The prediction for the receptor, based on free web SOPMA calculations, is 43 alpha-helix. The agreement of observation and expectation is evidence for a co.Lot showed inclusion body (IB) and membrane fractions (M) of OPRM. doi:10.1371/journal.pone.0056500.gConfirmation of Full Length of OPRMOPRM, western blot positive for the N-terminal his-tag, was found at a position of around 38 kDa on 12 SDS-PAGE (Figure 4), though the expected Mw is 46 kDa. Several integral membrane proteins including several GPCRs were found to migrate anomalously smaller than expected on SDS AGE due toOPRM from E. coliFigure 2. Growth conditions of OPRM in different E.coli strains. Expression of OPRM was induced by IPTG. Cell culture density (OD600) and weight of cell pellet (g) after different induction times with two different media (TB and DYT) was measured. Cell pellet (g) was obtained from 1 liter of culture medium. doi:10.1371/journal.pone.0056500.gtheir hydrophobicity and compact structure [30]. Nevertheless, the presence of the full-length protein had to be confirmed. The protein was extracted from SDS-PAGE, digested with trypsin and treated with iodoacetamide and DTT for analysis by mass spectrometry. Only after treatment with DTT and iodoacetamide before digestion with trypsin peptide matches were found (Figure 6A). Four matches were further analyzed by MS/ MS analysis. These peptides were derived from cytoplasmic and intracellular loops connecting transmembrane domains, but not from the N-terminal domain that does not contain a trypsin cleavage site. A total of 13 sequence coverage was obtained (Figure 6B). As the C-terminal peptide was also found, the band with apparent Mw of 38 kDa in SDS-PAGE corresponded to the full length of the 46 kDa protein.isolated in Peak 1 (Figure 5) was found to have an alpha-helical content corresponding to 5? TM-helices (data not shown).Confirmation of Receptor Function by Agonist BindingThe functionality of the isolated OPRM was probed by measuring the binding of the natural ligand endomorphine-1 to OPRM by plasmon surface resonance. Initially about 8000 RUs of OPRM (MW 46 kDa) were bound to the Ni-NTA chip. After extensively washing with buffer ca. 4000 RU remained. These results illustrated that for membrane proteins high initial responses may be observed because of unspecific binding or aggregation. The addition of reducing agent (1 mM TCEP) to the loading buffer did not change the binding of OPRM. Upon supplying increasing concentrations of agonist EM-1 to the immobilized OPRM increasing binding signal (RU) was observed (Rmax = 40 RU (EM-1: MW 610 Da)). Evaluation with a 1:1 interaction model allowed determining a KD of 61618 nM for the binding of EM-1 to OPRM isolated in detergent FOS-12 (Figure 8), which confirmed the agonist binding capacity of the isolated OPRM. No binding of endomorphine-1 was observed for reduced OPRM, which was immobilized on the chip in 1 mM TCEP. This negative control indicated that the endomorphine-1 binding pocket was stabilized by a disulfide-bond in OPRM.Confirmation of 7-TM Alpha-helical Secondary StructureA first 12926553 characterization of OPRM receptor natively purified from bacterial membrane was carried out by circular dichroism. The secondary structure of the purified OPRM after gel filtration was determined by CD-data from the far-UV spectrum in the 200?50 nm range (Figure 7) by K2D deconvolution. The folded protein was characterized to have a secondary structure of 4665 alpha-helix. The prediction for the receptor, based on free web SOPMA calculations, is 43 alpha-helix. The agreement of observation and expectation is evidence for a co.Lot showed inclusion body (IB) and membrane fractions (M) of OPRM. doi:10.1371/journal.pone.0056500.gConfirmation of Full Length of OPRMOPRM, western blot positive for the N-terminal his-tag, was found at a position of around 38 kDa on 12 SDS-PAGE (Figure 4), though the expected Mw is 46 kDa. Several integral membrane proteins including several GPCRs were found to migrate anomalously smaller than expected on SDS AGE due toOPRM from E. coliFigure 2. Growth conditions of OPRM in different E.coli strains. Expression of OPRM was induced by IPTG. Cell culture density (OD600) and weight of cell pellet (g) after different induction times with two different media (TB and DYT) was measured. Cell pellet (g) was obtained from 1 liter of culture medium. doi:10.1371/journal.pone.0056500.gtheir hydrophobicity and compact structure [30]. Nevertheless, the presence of the full-length protein had to be confirmed. The protein was extracted from SDS-PAGE, digested with trypsin and treated with iodoacetamide and DTT for analysis by mass spectrometry. Only after treatment with DTT and iodoacetamide before digestion with trypsin peptide matches were found (Figure 6A). Four matches were further analyzed by MS/ MS analysis. These peptides were derived from cytoplasmic and intracellular loops connecting transmembrane domains, but not from the N-terminal domain that does not contain a trypsin cleavage site. A total of 13 sequence coverage was obtained (Figure 6B). As the C-terminal peptide was also found, the band with apparent Mw of 38 kDa in SDS-PAGE corresponded to the full length of the 46 kDa protein.isolated in Peak 1 (Figure 5) was found to have an alpha-helical content corresponding to 5? TM-helices (data not shown).Confirmation of Receptor Function by Agonist BindingThe functionality of the isolated OPRM was probed by measuring the binding of the natural ligand endomorphine-1 to OPRM by plasmon surface resonance. Initially about 8000 RUs of OPRM (MW 46 kDa) were bound to the Ni-NTA chip. After extensively washing with buffer ca. 4000 RU remained. These results illustrated that for membrane proteins high initial responses may be observed because of unspecific binding or aggregation. The addition of reducing agent (1 mM TCEP) to the loading buffer did not change the binding of OPRM. Upon supplying increasing concentrations of agonist EM-1 to the immobilized OPRM increasing binding signal (RU) was observed (Rmax = 40 RU (EM-1: MW 610 Da)). Evaluation with a 1:1 interaction model allowed determining a KD of 61618 nM for the binding of EM-1 to OPRM isolated in detergent FOS-12 (Figure 8), which confirmed the agonist binding capacity of the isolated OPRM. No binding of endomorphine-1 was observed for reduced OPRM, which was immobilized on the chip in 1 mM TCEP. This negative control indicated that the endomorphine-1 binding pocket was stabilized by a disulfide-bond in OPRM.Confirmation of 7-TM Alpha-helical Secondary StructureA first 12926553 characterization of OPRM receptor natively purified from bacterial membrane was carried out by circular dichroism. The secondary structure of the purified OPRM after gel filtration was determined by CD-data from the far-UV spectrum in the 200?50 nm range (Figure 7) by K2D deconvolution. The folded protein was characterized to have a secondary structure of 4665 alpha-helix. The prediction for the receptor, based on free web SOPMA calculations, is 43 alpha-helix. The agreement of observation and expectation is evidence for a co.Lot showed inclusion body (IB) and membrane fractions (M) of OPRM. doi:10.1371/journal.pone.0056500.gConfirmation of Full Length of OPRMOPRM, western blot positive for the N-terminal his-tag, was found at a position of around 38 kDa on 12 SDS-PAGE (Figure 4), though the expected Mw is 46 kDa. Several integral membrane proteins including several GPCRs were found to migrate anomalously smaller than expected on SDS AGE due toOPRM from E. coliFigure 2. Growth conditions of OPRM in different E.coli strains. Expression of OPRM was induced by IPTG. Cell culture density (OD600) and weight of cell pellet (g) after different induction times with two different media (TB and DYT) was measured. Cell pellet (g) was obtained from 1 liter of culture medium. doi:10.1371/journal.pone.0056500.gtheir hydrophobicity and compact structure [30]. Nevertheless, the presence of the full-length protein had to be confirmed. The protein was extracted from SDS-PAGE, digested with trypsin and treated with iodoacetamide and DTT for analysis by mass spectrometry. Only after treatment with DTT and iodoacetamide before digestion with trypsin peptide matches were found (Figure 6A). Four matches were further analyzed by MS/ MS analysis. These peptides were derived from cytoplasmic and intracellular loops connecting transmembrane domains, but not from the N-terminal domain that does not contain a trypsin cleavage site. A total of 13 sequence coverage was obtained (Figure 6B). As the C-terminal peptide was also found, the band with apparent Mw of 38 kDa in SDS-PAGE corresponded to the full length of the 46 kDa protein.isolated in Peak 1 (Figure 5) was found to have an alpha-helical content corresponding to 5? TM-helices (data not shown).Confirmation of Receptor Function by Agonist BindingThe functionality of the isolated OPRM was probed by measuring the binding of the natural ligand endomorphine-1 to OPRM by plasmon surface resonance. Initially about 8000 RUs of OPRM (MW 46 kDa) were bound to the Ni-NTA chip. After extensively washing with buffer ca. 4000 RU remained. These results illustrated that for membrane proteins high initial responses may be observed because of unspecific binding or aggregation. The addition of reducing agent (1 mM TCEP) to the loading buffer did not change the binding of OPRM. Upon supplying increasing concentrations of agonist EM-1 to the immobilized OPRM increasing binding signal (RU) was observed (Rmax = 40 RU (EM-1: MW 610 Da)). Evaluation with a 1:1 interaction model allowed determining a KD of 61618 nM for the binding of EM-1 to OPRM isolated in detergent FOS-12 (Figure 8), which confirmed the agonist binding capacity of the isolated OPRM. No binding of endomorphine-1 was observed for reduced OPRM, which was immobilized on the chip in 1 mM TCEP. This negative control indicated that the endomorphine-1 binding pocket was stabilized by a disulfide-bond in OPRM.Confirmation of 7-TM Alpha-helical Secondary StructureA first 12926553 characterization of OPRM receptor natively purified from bacterial membrane was carried out by circular dichroism. The secondary structure of the purified OPRM after gel filtration was determined by CD-data from the far-UV spectrum in the 200?50 nm range (Figure 7) by K2D deconvolution. The folded protein was characterized to have a secondary structure of 4665 alpha-helix. The prediction for the receptor, based on free web SOPMA calculations, is 43 alpha-helix. The agreement of observation and expectation is evidence for a co.