ABSTRACT
Novel benzofuran-2-carboxamide ligands, which are selective for sigma receptors, have been synthesized via a microwave-assisted Perkin rearrangement reaction and a modified Finkelstein halogen-exchange used to facilitate N-alkylation. The ligands synthesized are the 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamides (KSCM-1, KSCM-5 and KSCM-11). The benzofuran-2-carboxamide structure was N-arylated and N-alkylated to include both N-phenyl and N-(3-(piperidin-1-yl)propyl substituents, respectively. These new carboxamides exhibit high affinity at the sigma-1 receptor with K(i) values ranging from 7.8 to 34nM. Ligand KSCM-1 with two methoxy substituents at C-5 and C-6 of the benzofuran ring, and K(i)=27.5nM at sigma-1 was found to be more selective for sigma-1 over sigma-2.
Subject(s)
Benzofurans/chemistry , Benzofurans/pharmacology , Receptors, sigma/metabolism , Animals , Benzofurans/chemical synthesis , Cell Line , Humans , Ligands , Radioligand Assay , Receptors, sigma/antagonists & inhibitors , Structure-Activity Relationship , Sigma-1 ReceptorABSTRACT
Recent advances in technology have allowed for the identification of complex protein mixtures in a rapid fashion. This report highlights the use of 2D gel electrophoresis, mass spectrometry, and database analysis to determine contaminating species of the Escherichia coli genome that are present during immobilized metal affinity chromatography (IMAC), highlighting Co(2+) as the affinity ligand. Four proteins (triosephosphate isomerase, alpha galactosidase, Hsp90, and glucosamine 6-phosphate synthase) constitute the majority of E. coli proteins that bind and potentially may coelute during chromatography. Results are discussed within the context of changes that when implemented could lead to an increase in IMAC efficiency, not by altering column conditions, but rather by changing the nature of the nuisance proteins that principally reduce column capacity and extend processing times. Such a study illustrates the use of proteome data to aid in bioprocess design.
Subject(s)
Amino Acid Sequence , Chromatography, Affinity/methods , Cobalt , Databases, Genetic , Escherichia coli/genetics , Proteomics/methods , Chelating Agents , Electrophoresis, Gel, Two-Dimensional , Mass Spectrometry , Metals , Molecular Sequence Data , Protein Engineering , Proteins/genetics , Species SpecificityABSTRACT
The chloroplast signal recognition particle consists of a conserved 54-kDa GTPase and a novel 43-kDa chromodomain protein (cpSRP43) that together bind light-harvesting chlorophyll a/b-binding protein (LHCP) to form a soluble targeting complex that is subsequently directed to the thylakoid membrane. Homology-based modeling of cpSRP43 indicates the presence of two previously identified chromodomains along with a third N-terminal chromodomain. Chromodomain deletion constructs were used to examine the role of each chromodomain in mediating distinct steps in the LHCP localization mechanism. The C-terminal chromodomain is completely dispensable for LHCP targeting/integration in vitro. The central chromodomain is essential for both targeting complex formation and integration because of its role in binding the M domain of cpSRP54. The N-terminal chromodomain (CD1) is unnecessary for targeting complex formation but is required for integration. This correlates with the ability of CD1 along with the ankyrin repeat region of cpSRP43 to regulate the GTPase cycle of the cpSRP-receptor complex.
Subject(s)
GTP Phosphohydrolases/chemistry , Signal Recognition Particle/physiology , Amino Acid Sequence , Ankyrins/chemistry , Apoproteins/chemistry , Arabidopsis , Biological Transport , Chloroplast Proteins , Chloroplasts/chemistry , Chloroplasts/metabolism , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Endoplasmic Reticulum/metabolism , Gene Deletion , Glutathione Transferase/metabolism , Hydrolysis , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Photosystem II Protein Complex/chemistry , Plant Proteins/chemistry , Protein Biosynthesis , Protein Processing, Post-Translational , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Ribosomes/chemistry , Signal Recognition Particle/chemistry , Signal Transduction , Thylakoids/metabolism , Two-Hybrid System TechniquesABSTRACT
Integration of thylakoid proteins by the chloroplast signal recognition particle (cpSRP) posttranslational transport pathway requires the cpSRP, an SRP receptor homologue (cpFtsY), and the membrane protein ALB3. Similarly, Escherichia coli uses an SRP and FtsY to cotranslationally target membrane proteins to the SecYEG translocase, which contains an ALB3 homologue, YidC. In neither system are the interactions between soluble and membrane components well understood. We show that complexes containing cpSRP, cpFtsY, and ALB3 can be precipitated using affinity tags on cpSRP or cpFtsY. Stabilization of this complex with GMP-PNP specifically blocks subsequent integration of substrate (light harvesting chl a/b-binding protein [LHCP]), indicating that the complex occupies functional ALB3 translocation sites. Surprisingly, neither substrate nor cpSRP43, a component of cpSRP, was necessary to form a complex with ALB3. Complexes also contained cpSecY, but its removal did not inhibit ALB3 function. Furthermore, antibody bound to ALB3 prevented ALB3 association with cpSRP and cpFtsY and inhibited LHCP integration suggesting that a complex containing cpSRP, cpFtsY, and ALB3 must form for proper LHCP integration.
Subject(s)
Arabidopsis Proteins/metabolism , Bacterial Proteins/metabolism , Chloroplasts/enzymology , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Recognition Particle/metabolism , Thylakoids/enzymology , Arabidopsis , Bacterial Proteins/genetics , Chloroplast Proteins , Receptors, Cytoplasmic and Nuclear/genetics , Signal Recognition Particle/genetics , Substrate SpecificityABSTRACT
The signal recognition particle (SRP) and its receptor (FtsY in prokaryotes) are essential for cotranslational protein targeting to the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in prokaryotes. An SRP/FtsY-like protein targeting/integration pathway in chloroplasts mediates the posttranslational integration of the light-harvesting chlorophyll a/b-binding protein (LHCP) into thylakoid membranes. GTP, chloroplast SRP (cpSRP), and chloroplast FtsY (cpFtsY) are required for LHCP integration into thylakoid membranes. Here, we report the reconstitution of the LHCP integration reaction with purified recombinant proteins and salt-washed thylakoids. Our data demonstrate that cpSRP and cpFtsY are the only soluble protein components required for LHCP integration. In addition, our studies reveal that ATP, though not absolutely required, remarkably stimulates LHCP integration into salt-washed thylakoids. ATP stimulates LHCP integration by a mechanism independent of the thylakoidal pH gradient (DeltapH) and exerts no detectable effect on the formation of the soluble LHCP-cpSRP-targeting complex. Taken together, our results indicate the participation of a thylakoid ATP-binding protein in LHCP integration.
Subject(s)
Adenosine Triphosphate/metabolism , Arabidopsis/metabolism , Bacterial Proteins/metabolism , Chloroplasts/metabolism , Photosynthetic Reaction Center Complex Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Recognition Particle/metabolism , Arabidopsis/genetics , Bacterial Proteins/genetics , Light-Harvesting Protein Complexes , Plasmids , Protein Sorting Signals/physiology , Receptors, Cytoplasmic and Nuclear/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Thylakoids/metabolismABSTRACT
A new component of the bacterial translocation machinery, YidC, has been identified that specializes in the integration of membrane proteins. YidC is homologous to the mitochondrial Oxa1p and the chloroplast Alb3, which functions in a novel pathway for the insertion of membrane proteins from the mitochondrial matrix and chloroplast stroma, respectively. We find that Alb3 can functionally complement the Escherichia coli YidC depletion strain and promote the membrane insertion of the M13 procoat and leader peptidase that were previously shown to depend on the bacterial YidC for membrane translocation. In addition, the chloroplast Alb3 that is expressed in bacteria is essential for the insertion of chloroplast cpSecE protein into the bacterial inner membrane. Surprisingly, Alb3 is not required for the insertion of cpSecE into the thylakoid membrane. These results underscore the importance of Oxa1p homologs for membrane protein insertion in bacteria and demonstrate that the requirement for Oxa1p homologs is different in the bacterial and thylakoid membrane systems.
