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Isolation of thylakoid membrane complexes from rice by a new double-strips BN/SDS-PAGE and bioinformatics prediction of stromal ridge subunits interaction.

Shao J, Zhang Y, Yu J, Guo L, Ding Y - PLoS ONE (2011)

Bottom Line: The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp).The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice.This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Hybrid Rice, Department of Genetics, College of Life Sciences, Wuhan University, Wuhan, China.

ABSTRACT
Thylakoid membrane complexes of rice (Oryza sativa L.) play crucial roles in growth and crop production. Understanding of protein interactions within the complex would provide new insights into photosynthesis. Here, a new "Double-Strips BN/SDS-PAGE" method was employed to separate thylakoid membrane complexes in order to increase the protein abundance on 2D-gels and to facilitate the identification of hydrophobic transmembrane proteins. A total of 58 protein spots could be observed and subunit constitution of these complexes exhibited on 2D-gels. The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp). Furthermore, the proteins separated from rice thylakoid membrane were identified by the mass spectrometry (MS). The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice. Using molecular dynamics simulation to explore the recognition mechanism of these subunits, we showed that salt bridge interactions between residues R19 of PsaC and E168 of PasD as well as R75 of PsaC and E91 of PsaD played important roles in the stability of the complex. This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy.

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Related in: MedlinePlus

The operation of a novel “Double-Strips 2D BN/SDS-PAGE” transfer technique from the first dimension to the second.A: One strip of 1D BN-gel lane was placed in the glass plate after denaturing. B: The other identical BN-gel strip was covered to the previous according to the aligned protein complexes, and then the glass plate was overlapped on the gels. C: The cassette was placed in skew form avoiding air bubble produced, the agarose was poured in the space between the BN-gel and stacking gel (just the half of all volume, as arrow indicated) after acrylamide was polymerized. D: The remaining space was subsequently filled with agarose until the previous solution has been concreted.
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pone-0020342-g002: The operation of a novel “Double-Strips 2D BN/SDS-PAGE” transfer technique from the first dimension to the second.A: One strip of 1D BN-gel lane was placed in the glass plate after denaturing. B: The other identical BN-gel strip was covered to the previous according to the aligned protein complexes, and then the glass plate was overlapped on the gels. C: The cassette was placed in skew form avoiding air bubble produced, the agarose was poured in the space between the BN-gel and stacking gel (just the half of all volume, as arrow indicated) after acrylamide was polymerized. D: The remaining space was subsequently filled with agarose until the previous solution has been concreted.

Mentions: In the traditional BN/SDS-PAGE method (e.g. single strip BN/SDS-PAGE), the amount of sample loaded in 1D BN-gels was limited by the slot volume, and furthermore, just only one lane strip was transferred to the second dimensional, some constituent subunits of the membrane complexes, especially for those expressed in low level, could not be exhibited on 2D SDS-gels. In order to circumvent the obstacle, a new gel-based method “Double-Strips BN/SDS-PAGE” was developed in this study for the purpose of increasing the abundance of protein spots separated in 2D SDS-gels. The main workflow of this method was described in the method section and illustrated in Figure 2.


Isolation of thylakoid membrane complexes from rice by a new double-strips BN/SDS-PAGE and bioinformatics prediction of stromal ridge subunits interaction.

Shao J, Zhang Y, Yu J, Guo L, Ding Y - PLoS ONE (2011)

The operation of a novel “Double-Strips 2D BN/SDS-PAGE” transfer technique from the first dimension to the second.A: One strip of 1D BN-gel lane was placed in the glass plate after denaturing. B: The other identical BN-gel strip was covered to the previous according to the aligned protein complexes, and then the glass plate was overlapped on the gels. C: The cassette was placed in skew form avoiding air bubble produced, the agarose was poured in the space between the BN-gel and stacking gel (just the half of all volume, as arrow indicated) after acrylamide was polymerized. D: The remaining space was subsequently filled with agarose until the previous solution has been concreted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102703&req=5

pone-0020342-g002: The operation of a novel “Double-Strips 2D BN/SDS-PAGE” transfer technique from the first dimension to the second.A: One strip of 1D BN-gel lane was placed in the glass plate after denaturing. B: The other identical BN-gel strip was covered to the previous according to the aligned protein complexes, and then the glass plate was overlapped on the gels. C: The cassette was placed in skew form avoiding air bubble produced, the agarose was poured in the space between the BN-gel and stacking gel (just the half of all volume, as arrow indicated) after acrylamide was polymerized. D: The remaining space was subsequently filled with agarose until the previous solution has been concreted.
Mentions: In the traditional BN/SDS-PAGE method (e.g. single strip BN/SDS-PAGE), the amount of sample loaded in 1D BN-gels was limited by the slot volume, and furthermore, just only one lane strip was transferred to the second dimensional, some constituent subunits of the membrane complexes, especially for those expressed in low level, could not be exhibited on 2D SDS-gels. In order to circumvent the obstacle, a new gel-based method “Double-Strips BN/SDS-PAGE” was developed in this study for the purpose of increasing the abundance of protein spots separated in 2D SDS-gels. The main workflow of this method was described in the method section and illustrated in Figure 2.

Bottom Line: The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp).The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice.This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Hybrid Rice, Department of Genetics, College of Life Sciences, Wuhan University, Wuhan, China.

ABSTRACT
Thylakoid membrane complexes of rice (Oryza sativa L.) play crucial roles in growth and crop production. Understanding of protein interactions within the complex would provide new insights into photosynthesis. Here, a new "Double-Strips BN/SDS-PAGE" method was employed to separate thylakoid membrane complexes in order to increase the protein abundance on 2D-gels and to facilitate the identification of hydrophobic transmembrane proteins. A total of 58 protein spots could be observed and subunit constitution of these complexes exhibited on 2D-gels. The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp). Furthermore, the proteins separated from rice thylakoid membrane were identified by the mass spectrometry (MS). The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice. Using molecular dynamics simulation to explore the recognition mechanism of these subunits, we showed that salt bridge interactions between residues R19 of PsaC and E168 of PasD as well as R75 of PsaC and E91 of PsaD played important roles in the stability of the complex. This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy.

Show MeSH
Related in: MedlinePlus