Nanao Suzuki | St Jude Childrens research Hospital (original) (raw)

Papers by Nanao Suzuki

Research paper thumbnail of Calcium-dependent structural changes in human reticulocalbin-1

Journal of Biochemistry, 2014

Research paper thumbnail of An efficient screening method for purifying and crystallizing membrane proteins using modified clear-native PAGE

Analytical Biochemistry, 2018

Membrane proteins, such as G-protein coupled receptors, control communication between cells and t... more Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming and trial-and-error process. Here, we report a rapid method for precrystallization screening of membrane proteins using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with the modified Coomassie Brilliant Blue G-250 (mCBB) stain that was reduced in sodium formate. A 2A adenosine receptor (A 2A AR) was selected as a target membrane protein, for which we previously obtained the crystal structure using an antibody, and was expressed as a red fluorescent protein fusion for in-gel fluorescence detection. The mCBB CN-PAGE method enabled the optimization of the solubilization, purification, and crystallization conditions of A 2A AR using the solubilized membrane fraction expressing the protein without purification procedures. These data suggest the applicability of mCBB CN-PAGE technique to a wide variety of integral membrane proteins.

Research paper thumbnail of Coordination structures of Mg 2+ and Ca 2+ in three types of tobacco calmodulins in solution: Fourier-transform infrared spectroscopic studies of side-chain COO − groups

Biopolymers, 2013

Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular act... more Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg(2+) and Ca(2+) among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak β-strand bands and the weak 1661 cm(-1) band. Coordination structures of Mg(2+) of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca(2+) -binding manner was similar among the three CaMs. The amplitude differences of the band at 1554-1550 cm(-1) obtained by second-derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca(2+) ] increases under low [Ca(2+) ] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca(2+) ]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg(2+) -binding manner and needs higher [Ca(2+) ] for bidentate Ca(2+) coordination of 12th Glu in EF-hand motifs. The Ca(2+) -binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation-dependent conformational change in NtCaM13 is unique among the three NtCaMs. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 472-483, 2013.

Research paper thumbnail of Coordination structures of Mg 2+ and Ca 2+ in three types of tobacco calmodulins in solution: Fourier-transform infrared spectroscopic studies of side-chain COO − groups

Biopolymers, 2013

Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular act... more Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg(2+) and Ca(2+) among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak β-strand bands and the weak 1661 cm(-1) band. Coordination structures of Mg(2+) of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca(2+) -binding manner was similar among the three CaMs. The amplitude differences of the band at 1554-1550 cm(-1) obtained by second-derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca(2+) ] increases under low [Ca(2+) ] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca(2+) ]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg(2+) -binding manner and needs higher [Ca(2+) ] for bidentate Ca(2+) coordination of 12th Glu in EF-hand motifs. The Ca(2+) -binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation-dependent conformational change in NtCaM13 is unique among the three NtCaMs. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 472-483, 2013.

Research paper thumbnail of Coordination structures of Mg 2+ and Ca 2+ in three types of tobacco calmodulins in solution: Fourier-transform infrared spectroscopic studies of side-chain COO − groups

Biopolymers, 2013

Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular act... more Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg(2+) and Ca(2+) among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak β-strand bands and the weak 1661 cm(-1) band. Coordination structures of Mg(2+) of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca(2+) -binding manner was similar among the three CaMs. The amplitude differences of the band at 1554-1550 cm(-1) obtained by second-derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca(2+) ] increases under low [Ca(2+) ] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca(2+) ]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg(2+) -binding manner and needs higher [Ca(2+) ] for bidentate Ca(2+) coordination of 12th Glu in EF-hand motifs. The Ca(2+) -binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation-dependent conformational change in NtCaM13 is unique among the three NtCaMs. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 472-483, 2013.

Research paper thumbnail of An efficient screening method for purifying and crystallizing membrane proteins using modified clear-native PAGE

Anal Biochem., 2018

Membrane proteins, such as G-protein coupled receptors, control communication between cells and t... more Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming and trial-and-error process. Here, we report a rapid method for pre-crystallization screening of membrane proteins using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with the modified Coomassie Brilliant Blue G-250 (mCBB) stain that was reduced in sodium formate. A 2A adenosine receptor (A 2A AR) was selected as a target membrane protein, for which we previously obtained the crystal structure using an antibody, and was expressed as a red fluorescent protein fusion for in-gel fluorescence detection. The mCBB CN-PAGE method enabled the optimization of the solubilization, purification, and crys-tallization conditions of A 2A AR using the solubilized membrane fraction expressing the protein without purification procedures. These data suggest the applicability of mCBB CN-PAGE technique to a wide variety of integral membrane proteins.

Research paper thumbnail of Calcium-dependent structural changes in human reticulocalbin-1.

J Biochem., 2014

Human reticulocalbin-1 (hRCN1) has six EF-hand motifs and binds Ca(2+). hRCN1 is a member of the ... more Human reticulocalbin-1 (hRCN1) has six EF-hand motifs and binds Ca(2+). hRCN1 is a member of the CREC family localized in the secretory pathway, and its cellular function remains unclear. In this study, we established a new bacterial expression and purification procedure for hRCN1. We observed that hRCN1 binds Ca(2+) in a cooperative manner and the Ca(2+) binding caused an increase in the α-helix content of hRCN1. On the other hand, hRCN1 did not change the structure with Mg(2+) loading. hRCN1 is a monomeric protein, and its overall structure became more compact upon Ca(2+) binding, as revealed by gel-filtration column chromatography and small-angle X-ray scattering. This is the first report of conformational changes in the CREC family upon Ca(2+) binding. Our data suggest that CREC family member interactions with target proteins are regulated in the secretory pathway by conformational changes upon Ca(2+) binding.

Research paper thumbnail of Spectroscopic analysis of three types of tobacco calmodulins at different Ca'2+'concentrations and pHs

Biopolymers., Jan 1, 2013

Calmodulin (CaM) is a Ca(2+)-binding protein that regulates a number of fundamental cellular acti... more Calmodulin (CaM) is a Ca(2+)-binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg(2+) and Ca(2+) among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak β-strand bands and the weak 1661 cm(-1) band. Coordination structures of Mg(2+) of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca(2+)-binding manner was similar among the three CaMs. The amplitude differences of the band at 1554-1550 cm(-1) obtained by second-derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca(2+)] increases under low [Ca(2+)] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca(2+)]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg(2+)-binding manner and needs higher [Ca(2+)] for bidentate Ca(2+) coordination of 12th Glu in EF-hand motifs. The Ca(2+)-binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation-dependent conformational change in NtCaM13 is unique among the three NtCaMs.

Research paper thumbnail of Calcium-dependent structural changes in human reticulocalbin-1

Journal of Biochemistry, 2014

Research paper thumbnail of An efficient screening method for purifying and crystallizing membrane proteins using modified clear-native PAGE

Analytical Biochemistry, 2018

Membrane proteins, such as G-protein coupled receptors, control communication between cells and t... more Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming and trial-and-error process. Here, we report a rapid method for precrystallization screening of membrane proteins using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with the modified Coomassie Brilliant Blue G-250 (mCBB) stain that was reduced in sodium formate. A 2A adenosine receptor (A 2A AR) was selected as a target membrane protein, for which we previously obtained the crystal structure using an antibody, and was expressed as a red fluorescent protein fusion for in-gel fluorescence detection. The mCBB CN-PAGE method enabled the optimization of the solubilization, purification, and crystallization conditions of A 2A AR using the solubilized membrane fraction expressing the protein without purification procedures. These data suggest the applicability of mCBB CN-PAGE technique to a wide variety of integral membrane proteins.

Research paper thumbnail of Coordination structures of Mg 2+ and Ca 2+ in three types of tobacco calmodulins in solution: Fourier-transform infrared spectroscopic studies of side-chain COO − groups

Biopolymers, 2013

Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular act... more Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg(2+) and Ca(2+) among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak β-strand bands and the weak 1661 cm(-1) band. Coordination structures of Mg(2+) of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca(2+) -binding manner was similar among the three CaMs. The amplitude differences of the band at 1554-1550 cm(-1) obtained by second-derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca(2+) ] increases under low [Ca(2+) ] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca(2+) ]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg(2+) -binding manner and needs higher [Ca(2+) ] for bidentate Ca(2+) coordination of 12th Glu in EF-hand motifs. The Ca(2+) -binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation-dependent conformational change in NtCaM13 is unique among the three NtCaMs. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 472-483, 2013.

Research paper thumbnail of Coordination structures of Mg 2+ and Ca 2+ in three types of tobacco calmodulins in solution: Fourier-transform infrared spectroscopic studies of side-chain COO − groups

Biopolymers, 2013

Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular act... more Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg(2+) and Ca(2+) among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak β-strand bands and the weak 1661 cm(-1) band. Coordination structures of Mg(2+) of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca(2+) -binding manner was similar among the three CaMs. The amplitude differences of the band at 1554-1550 cm(-1) obtained by second-derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca(2+) ] increases under low [Ca(2+) ] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca(2+) ]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg(2+) -binding manner and needs higher [Ca(2+) ] for bidentate Ca(2+) coordination of 12th Glu in EF-hand motifs. The Ca(2+) -binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation-dependent conformational change in NtCaM13 is unique among the three NtCaMs. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 472-483, 2013.

Research paper thumbnail of Coordination structures of Mg 2+ and Ca 2+ in three types of tobacco calmodulins in solution: Fourier-transform infrared spectroscopic studies of side-chain COO − groups

Biopolymers, 2013

Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular act... more Calmodulin (CaM) is a Ca(2+) -binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg(2+) and Ca(2+) among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak β-strand bands and the weak 1661 cm(-1) band. Coordination structures of Mg(2+) of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca(2+) -binding manner was similar among the three CaMs. The amplitude differences of the band at 1554-1550 cm(-1) obtained by second-derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca(2+) ] increases under low [Ca(2+) ] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca(2+) ]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg(2+) -binding manner and needs higher [Ca(2+) ] for bidentate Ca(2+) coordination of 12th Glu in EF-hand motifs. The Ca(2+) -binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation-dependent conformational change in NtCaM13 is unique among the three NtCaMs. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 472-483, 2013.

Research paper thumbnail of An efficient screening method for purifying and crystallizing membrane proteins using modified clear-native PAGE

Anal Biochem., 2018

Membrane proteins, such as G-protein coupled receptors, control communication between cells and t... more Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming and trial-and-error process. Here, we report a rapid method for pre-crystallization screening of membrane proteins using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with the modified Coomassie Brilliant Blue G-250 (mCBB) stain that was reduced in sodium formate. A 2A adenosine receptor (A 2A AR) was selected as a target membrane protein, for which we previously obtained the crystal structure using an antibody, and was expressed as a red fluorescent protein fusion for in-gel fluorescence detection. The mCBB CN-PAGE method enabled the optimization of the solubilization, purification, and crys-tallization conditions of A 2A AR using the solubilized membrane fraction expressing the protein without purification procedures. These data suggest the applicability of mCBB CN-PAGE technique to a wide variety of integral membrane proteins.

Research paper thumbnail of Calcium-dependent structural changes in human reticulocalbin-1.

J Biochem., 2014

Human reticulocalbin-1 (hRCN1) has six EF-hand motifs and binds Ca(2+). hRCN1 is a member of the ... more Human reticulocalbin-1 (hRCN1) has six EF-hand motifs and binds Ca(2+). hRCN1 is a member of the CREC family localized in the secretory pathway, and its cellular function remains unclear. In this study, we established a new bacterial expression and purification procedure for hRCN1. We observed that hRCN1 binds Ca(2+) in a cooperative manner and the Ca(2+) binding caused an increase in the α-helix content of hRCN1. On the other hand, hRCN1 did not change the structure with Mg(2+) loading. hRCN1 is a monomeric protein, and its overall structure became more compact upon Ca(2+) binding, as revealed by gel-filtration column chromatography and small-angle X-ray scattering. This is the first report of conformational changes in the CREC family upon Ca(2+) binding. Our data suggest that CREC family member interactions with target proteins are regulated in the secretory pathway by conformational changes upon Ca(2+) binding.

Research paper thumbnail of Spectroscopic analysis of three types of tobacco calmodulins at different Ca'2+'concentrations and pHs

Biopolymers., Jan 1, 2013

Calmodulin (CaM) is a Ca(2+)-binding protein that regulates a number of fundamental cellular acti... more Calmodulin (CaM) is a Ca(2+)-binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg(2+) and Ca(2+) among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak β-strand bands and the weak 1661 cm(-1) band. Coordination structures of Mg(2+) of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca(2+)-binding manner was similar among the three CaMs. The amplitude differences of the band at 1554-1550 cm(-1) obtained by second-derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca(2+)] increases under low [Ca(2+)] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca(2+)]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg(2+)-binding manner and needs higher [Ca(2+)] for bidentate Ca(2+) coordination of 12th Glu in EF-hand motifs. The Ca(2+)-binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation-dependent conformational change in NtCaM13 is unique among the three NtCaMs.