Rhodopsin oligomerization and dissociation in vivo and under experimental conditions is an important topic both for a basic understanding of photoreceptor structure-function but also as a potential eye disease mechani...Rhodopsin oligomerization and dissociation in vivo and under experimental conditions is an important topic both for a basic understanding of photoreceptor structure-function but also as a potential eye disease mechanism. In this study, to estimate a state rhodopsin after solubilization with mild and harsh detergents, we applied the native (blue native-PAGE, BN- PAGE) and denaturing electrophoresis (blue-urea- PAGE, BU-PAGE;blue-SDS-PAGE, BSDS-PAGE and SDS- PAGE). After blue BN-PAGE and BSDS- PAGE, rhodopsin and opsin, respectively, were presented in gels a major band of dimer with slight contents of higher oligomers without any traces of monomer, thus testifying in favor dimer-heteromeric state of frog rhodopsin in the photoreceptor membrane. Despite all oligomer bands gave positive staining with the rhodopsin-specific monoclonal antibodies (mAb), subsequent SDS-PAGE in combination with electroelution in denaturing conditions showed that stained bands are not homogenous and besides of opsin oli-gomers contain a small admixture of proteins with unknown function. Unfolding of opsin oligomers by solubilization in SDS, as compared with folded opsin in digitonin, induces their transition to a more compact conformation. It was manifested in a more rapid migration of opsin oligomers toward to anode. Cooling of digitonin/SDS mixed extracts at 4?C for 24 hours led to a partial reverse transition of unfolded opsin dimer to initial folded conformation, thus de- monstrating the entropic nature of this transition. Opsin monomer can be observed in the gels only after harsh dissociation of oligomers under BU-PAGE or SDS-PAGE. The electro elution of the individual opsin oligomers with denaturing buffer followed by SDS-PAGE resulted in dissociation of dimer to monomers. However, unexpectedly, the trimer was dissociated to a prevailing dimer and a small portion of monomer. The products dissociation of both opsin tetramer and pentamer are difficult to determine precisely, but they are neither monomer nor dimer. Dissociation data show that the degree of opsin oli- gomerization by unknown reasons affects the pattern of dissociation of its aggregates. Obtained in this paper data indicate a need for further detailed study the obscure mechanisms of aggregation-dissociation of rhodopsin.展开更多
Protein complexes are important for almost all biological processes.Hence,to fully understand how cells work,it is also necessary to characterize protein complexes and their dynamics in response to various cellular cu...Protein complexes are important for almost all biological processes.Hence,to fully understand how cells work,it is also necessary to characterize protein complexes and their dynamics in response to various cellular cues.Moreover,the dynamics of protein interaction play crucial roles in regulating the(dis)association of protein complexes and,in turn,regulating biological processes such as metabolism.Here,mitochondrial protein complexes were investigated by blue native PAGE and size-exclusion chromatography under conditions of oxidative stress in order to monitor their dynamic(dis)associations.Rearrangements of enzyme interactions and changes in protein complex abundance were observed in response to oxidative stress induced by menadione treatment.These included changes in enzymatic protein complexes involving g-amino butyric acid transaminase(GABA-T),D-ornithine aminotransferase(D-OAT),or proline dehydrogenase 1(POX1)that are expected to affect proline metabolism.Menadione treatment also affected interactions between several enzymes of the tricarboxylic acid(TCA)cycle and the abundance of complexes of the oxidative phosphorylation pathway.In addition,we compared the mitochondrial complexes of roots and shoots.Considerable differences between the two tissues were observed in the mitochondrial import/export apparatus,the formation of super-complexes in the oxidative phosphorylation pathway,and specific interactions between enzymes of the TCA cycle that we postulate may be related to the metabolic/energetic requirements of roots and shoots.展开更多
文摘Rhodopsin oligomerization and dissociation in vivo and under experimental conditions is an important topic both for a basic understanding of photoreceptor structure-function but also as a potential eye disease mechanism. In this study, to estimate a state rhodopsin after solubilization with mild and harsh detergents, we applied the native (blue native-PAGE, BN- PAGE) and denaturing electrophoresis (blue-urea- PAGE, BU-PAGE;blue-SDS-PAGE, BSDS-PAGE and SDS- PAGE). After blue BN-PAGE and BSDS- PAGE, rhodopsin and opsin, respectively, were presented in gels a major band of dimer with slight contents of higher oligomers without any traces of monomer, thus testifying in favor dimer-heteromeric state of frog rhodopsin in the photoreceptor membrane. Despite all oligomer bands gave positive staining with the rhodopsin-specific monoclonal antibodies (mAb), subsequent SDS-PAGE in combination with electroelution in denaturing conditions showed that stained bands are not homogenous and besides of opsin oli-gomers contain a small admixture of proteins with unknown function. Unfolding of opsin oligomers by solubilization in SDS, as compared with folded opsin in digitonin, induces their transition to a more compact conformation. It was manifested in a more rapid migration of opsin oligomers toward to anode. Cooling of digitonin/SDS mixed extracts at 4?C for 24 hours led to a partial reverse transition of unfolded opsin dimer to initial folded conformation, thus de- monstrating the entropic nature of this transition. Opsin monomer can be observed in the gels only after harsh dissociation of oligomers under BU-PAGE or SDS-PAGE. The electro elution of the individual opsin oligomers with denaturing buffer followed by SDS-PAGE resulted in dissociation of dimer to monomers. However, unexpectedly, the trimer was dissociated to a prevailing dimer and a small portion of monomer. The products dissociation of both opsin tetramer and pentamer are difficult to determine precisely, but they are neither monomer nor dimer. Dissociation data show that the degree of opsin oli- gomerization by unknown reasons affects the pattern of dissociation of its aggregates. Obtained in this paper data indicate a need for further detailed study the obscure mechanisms of aggregation-dissociation of rhodopsin.
基金supported by funding from the Max Planck Society(S.M.J.,A.G.,A.R.F.,and Y.Z.)the European Union’s Horizon 2020 research and innovation program,project PlantaSYST(SGA-CSA no.739582 under FPA no.664620)for supporting their researchfinancial support from the IMPRS-PMPG program.
文摘Protein complexes are important for almost all biological processes.Hence,to fully understand how cells work,it is also necessary to characterize protein complexes and their dynamics in response to various cellular cues.Moreover,the dynamics of protein interaction play crucial roles in regulating the(dis)association of protein complexes and,in turn,regulating biological processes such as metabolism.Here,mitochondrial protein complexes were investigated by blue native PAGE and size-exclusion chromatography under conditions of oxidative stress in order to monitor their dynamic(dis)associations.Rearrangements of enzyme interactions and changes in protein complex abundance were observed in response to oxidative stress induced by menadione treatment.These included changes in enzymatic protein complexes involving g-amino butyric acid transaminase(GABA-T),D-ornithine aminotransferase(D-OAT),or proline dehydrogenase 1(POX1)that are expected to affect proline metabolism.Menadione treatment also affected interactions between several enzymes of the tricarboxylic acid(TCA)cycle and the abundance of complexes of the oxidative phosphorylation pathway.In addition,we compared the mitochondrial complexes of roots and shoots.Considerable differences between the two tissues were observed in the mitochondrial import/export apparatus,the formation of super-complexes in the oxidative phosphorylation pathway,and specific interactions between enzymes of the TCA cycle that we postulate may be related to the metabolic/energetic requirements of roots and shoots.
