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Characterization and modeling of the oligomeric state and ligand binding behavior of purified translocator protein 18 kDa from Rhodobacter sphaeroides.

Li F, Xia Y, Meiler J, Ferguson-Miller S - Biochemistry (2013)

Bottom Line: Despite a number of TSPO ligands whose effects have been studied with respect to these varied biological activities, the nature of their interactions with TSPO and the molecular mechanism of their effects remain controversial, in part because of the lack of an atomic-resolution structure.Our results show that RsTSPO behaves as a dimer in the purified state and binds with low micromolar affinity to many of these ligands, including retinoic acid, curcumin, and a known Bcl-2 inhibitor, gossypol, suggesting a possible direct role for TSPO in their regulation of apoptosis.Binding behaviors of known ligands are discussed in the context of the model with respect to regions that may be involved in binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, United States.

ABSTRACT
Translocator Protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is a mitochondrial outer membrane protein that has been identified as a key player in cholesterol and porphyrin transport, apoptotic signaling, and cancer development, as well as neurological inflammation and disease. Despite a number of TSPO ligands whose effects have been studied with respect to these varied biological activities, the nature of their interactions with TSPO and the molecular mechanism of their effects remain controversial, in part because of the lack of an atomic-resolution structure. We expressed and purified the homologue of mammalian TSPO from Rhodobacter sphaeroides (RsTSPO), as well as a mutant form in a proposed drug binding loop, RsTSPOW38C. We characterized their binding behaviors with endogenous ligands and a series of compounds that affect apoptosis by using a sensitive tryptophan fluorescence quenching assay. Our results show that RsTSPO behaves as a dimer in the purified state and binds with low micromolar affinity to many of these ligands, including retinoic acid, curcumin, and a known Bcl-2 inhibitor, gossypol, suggesting a possible direct role for TSPO in their regulation of apoptosis. A computational model of the RsTSPO dimer is constructed using EM-Fold, Rosetta, and a cryo-electron microscopy density map. Binding behaviors of known ligands are discussed in the context of the model with respect to regions that may be involved in binding.

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Purificationand determination of the molecular mass of RsTSPO.(A) One representative trace of purified RsTSPO onthe triple detector array (Malvern). The peakof RsTSPO is labeled with a star, and the molecularmass was calculated as described and expressed on the Y axis. (B) SDS–PAGE for the purification of RsTSPO. The lane of SEC-purified RsTSPO is labeledwith a star corresponding to the light scattering profile. The trypsincutting mix represents the sample before SEC, while a purified RsTSPO10ht sample was used as the control.
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fig2: Purificationand determination of the molecular mass of RsTSPO.(A) One representative trace of purified RsTSPO onthe triple detector array (Malvern). The peakof RsTSPO is labeled with a star, and the molecularmass was calculated as described and expressed on the Y axis. (B) SDS–PAGE for the purification of RsTSPO. The lane of SEC-purified RsTSPO is labeledwith a star corresponding to the light scattering profile. The trypsincutting mix represents the sample before SEC, while a purified RsTSPO10ht sample was used as the control.

Mentions: RsTSPO was successfully purified to homogeneityby nickel affinity followed by size exclusion chromatography (SEC)(Figures 2 and 3A).The molecular mass of purified RsTSPO with a 10-histidinetag was determined to be 19371.76 ± 1.77 Da (17910.26 Da foruntagged RsTSPO) by mass spectrometry, correlatingwell with the calculated molecular mass from the amino acid sequence(19347.58 Da). The SEC running profile in 0.2% DM showed a peak thatsuggested a dimer form based on elution profile and molecular massstandards. To obtain a more accurate estimate of the size of RsTSPO, we characterized the purified RsTSPO in solution by using a combination of light scattering, UV,and refractive index measurements. A representative run of the UV280, refractive index, and light scattering measurements isshown in Figure 2A, and a representative SDS–PAGEgel is shown in Figure 2B. The molecular massof the RsTSPO–detergent complex was determinedto be ∼100 kDa from the scattering peak, with a protein fractionof ∼36% from comparison of scattering and 280 nm absorption;therefore, the molecular mass of the RsTSPO proteinin the complex was calculated to be 37 kDa, indicating a dimer of RsTSPO (given a monomer of 18 kDa) within the protein–detergentcomplex (Table 1). Purified ovalbumin and pureDM were also characterized under the same conditions. Ovalbumin showeda calculated molecular mass of ∼44 kDa as a monomer, and DMshowed a micelle size of ∼37 kDa; both agreed very well withliterature values and are consistent with the equivalent of approximatelytwo micelles of DM in the dimer–detergent complex.


Characterization and modeling of the oligomeric state and ligand binding behavior of purified translocator protein 18 kDa from Rhodobacter sphaeroides.

Li F, Xia Y, Meiler J, Ferguson-Miller S - Biochemistry (2013)

Purificationand determination of the molecular mass of RsTSPO.(A) One representative trace of purified RsTSPO onthe triple detector array (Malvern). The peakof RsTSPO is labeled with a star, and the molecularmass was calculated as described and expressed on the Y axis. (B) SDS–PAGE for the purification of RsTSPO. The lane of SEC-purified RsTSPO is labeledwith a star corresponding to the light scattering profile. The trypsincutting mix represents the sample before SEC, while a purified RsTSPO10ht sample was used as the control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756528&req=5

fig2: Purificationand determination of the molecular mass of RsTSPO.(A) One representative trace of purified RsTSPO onthe triple detector array (Malvern). The peakof RsTSPO is labeled with a star, and the molecularmass was calculated as described and expressed on the Y axis. (B) SDS–PAGE for the purification of RsTSPO. The lane of SEC-purified RsTSPO is labeledwith a star corresponding to the light scattering profile. The trypsincutting mix represents the sample before SEC, while a purified RsTSPO10ht sample was used as the control.
Mentions: RsTSPO was successfully purified to homogeneityby nickel affinity followed by size exclusion chromatography (SEC)(Figures 2 and 3A).The molecular mass of purified RsTSPO with a 10-histidinetag was determined to be 19371.76 ± 1.77 Da (17910.26 Da foruntagged RsTSPO) by mass spectrometry, correlatingwell with the calculated molecular mass from the amino acid sequence(19347.58 Da). The SEC running profile in 0.2% DM showed a peak thatsuggested a dimer form based on elution profile and molecular massstandards. To obtain a more accurate estimate of the size of RsTSPO, we characterized the purified RsTSPO in solution by using a combination of light scattering, UV,and refractive index measurements. A representative run of the UV280, refractive index, and light scattering measurements isshown in Figure 2A, and a representative SDS–PAGEgel is shown in Figure 2B. The molecular massof the RsTSPO–detergent complex was determinedto be ∼100 kDa from the scattering peak, with a protein fractionof ∼36% from comparison of scattering and 280 nm absorption;therefore, the molecular mass of the RsTSPO proteinin the complex was calculated to be 37 kDa, indicating a dimer of RsTSPO (given a monomer of 18 kDa) within the protein–detergentcomplex (Table 1). Purified ovalbumin and pureDM were also characterized under the same conditions. Ovalbumin showeda calculated molecular mass of ∼44 kDa as a monomer, and DMshowed a micelle size of ∼37 kDa; both agreed very well withliterature values and are consistent with the equivalent of approximatelytwo micelles of DM in the dimer–detergent complex.

Bottom Line: Despite a number of TSPO ligands whose effects have been studied with respect to these varied biological activities, the nature of their interactions with TSPO and the molecular mechanism of their effects remain controversial, in part because of the lack of an atomic-resolution structure.Our results show that RsTSPO behaves as a dimer in the purified state and binds with low micromolar affinity to many of these ligands, including retinoic acid, curcumin, and a known Bcl-2 inhibitor, gossypol, suggesting a possible direct role for TSPO in their regulation of apoptosis.Binding behaviors of known ligands are discussed in the context of the model with respect to regions that may be involved in binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, United States.

ABSTRACT
Translocator Protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is a mitochondrial outer membrane protein that has been identified as a key player in cholesterol and porphyrin transport, apoptotic signaling, and cancer development, as well as neurological inflammation and disease. Despite a number of TSPO ligands whose effects have been studied with respect to these varied biological activities, the nature of their interactions with TSPO and the molecular mechanism of their effects remain controversial, in part because of the lack of an atomic-resolution structure. We expressed and purified the homologue of mammalian TSPO from Rhodobacter sphaeroides (RsTSPO), as well as a mutant form in a proposed drug binding loop, RsTSPOW38C. We characterized their binding behaviors with endogenous ligands and a series of compounds that affect apoptosis by using a sensitive tryptophan fluorescence quenching assay. Our results show that RsTSPO behaves as a dimer in the purified state and binds with low micromolar affinity to many of these ligands, including retinoic acid, curcumin, and a known Bcl-2 inhibitor, gossypol, suggesting a possible direct role for TSPO in their regulation of apoptosis. A computational model of the RsTSPO dimer is constructed using EM-Fold, Rosetta, and a cryo-electron microscopy density map. Binding behaviors of known ligands are discussed in the context of the model with respect to regions that may be involved in binding.

Show MeSH
Related in: MedlinePlus