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1.
Biophys J ; 111(6): 1151-1162, 2016 Sep 20.
Article in English | MEDLINE | ID: mdl-27653474

ABSTRACT

Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico three-dimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs. Model selection was guided by single-molecule Förster resonance energy transfer experiments, which revealed the presence of at least two distinct conformations. Small angle x-ray scattering showed that the linking region among the GTPase (G-domain) and methionine-rich (M-domain) domains, an M-domain loop, and the cpSRP43 binding C-terminal extension of cpSRP54 are predominantly disordered. Interestingly, the linker and loop segments were observed to play an important role in organizing the domain arrangement of cpSRP54. Further, deletion of the finger loop abolished loading of the cpSRP cargo, light-harvesting chlorophyll binding protein. These data highlight important structural dynamics relevant to cpSRP54's role in the post- and cotranslational signaling processes.


Subject(s)
GTP Phosphohydrolases/chemistry , Signal Recognition Particle/chemistry , Animals , Arabidopsis , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Chloroplasts/metabolism , Dogs , Escherichia coli , Fluorescence Resonance Energy Transfer , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Methanocaldococcus , Molecular Dynamics Simulation , Mutation , Protein Domains , Scattering, Small Angle , Signal Recognition Particle/genetics , Signal Recognition Particle/metabolism , Structural Homology, Protein , Sulfolobus solfataricus , Thermus , X-Ray Diffraction
2.
J Biol Chem ; 290(25): 15462-15474, 2015 Jun 19.
Article in English | MEDLINE | ID: mdl-25918165

ABSTRACT

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Intracellular Membranes/metabolism , Signal Recognition Particle/metabolism , Thylakoids/metabolism , Amino Acid Motifs , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Intracellular Membranes/chemistry , Protein Binding/physiology , Protein Transport/physiology , Signal Recognition Particle/chemistry , Signal Recognition Particle/genetics , Thylakoids/chemistry , Thylakoids/genetics
3.
J Biol Chem ; 285(44): 34220-30, 2010 Oct 29.
Article in English | MEDLINE | ID: mdl-20729200

ABSTRACT

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP·LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.


Subject(s)
Arabidopsis Proteins/metabolism , Chloroplasts/metabolism , Signal Recognition Particle/metabolism , Cell Membrane/metabolism , Chloroplast Proteins , Cloning, Molecular , Guanosine Triphosphate/chemistry , Hydrolysis , Models, Biological , Pisum sativum/metabolism , Protein Binding , Protein Interaction Mapping , Protein Structure, Tertiary , Protein Transport , Recombinant Proteins/chemistry , Thylakoids/metabolism
4.
Protein Expr Purif ; 70(2): 191-5, 2010 Apr.
Article in English | MEDLINE | ID: mdl-19887109

ABSTRACT

The purpose of this study was to identify and characterize Escherichia coli proteins which display affinity towards both Immobilized Metal Affinity Chromatography (IMAC) and Hydrophobic Interaction Chromatography (HIC). Co(II) IMAC was chosen as the primary capture step, followed by HIC employing different concentrations of salt to promote adsorption. Results provided insight on this rather small pool of E. coli proteins. Nine out of the ten have isoelectric values less than six, and half are considered nonessential. These data indicate that the combination of IMAC and HIC could be developed as a potent method for the purification of recombinant proteins by judicious choice of the salt concentration used to promote HIC, the development of E. coli strain(s) deficient in certain genomic proteins, and the design of an IMAC-HIC affinity tail for recombinant protein isolation based on the very proteins deleted from the genome.


Subject(s)
Chromatography, Affinity/methods , Chromatography/methods , Escherichia coli Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Escherichia coli Proteins/isolation & purification , Hydrophobic and Hydrophilic Interactions , Protein Engineering
5.
J Biol Chem ; 284(22): 14891-903, 2009 May 29.
Article in English | MEDLINE | ID: mdl-19293157

ABSTRACT

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane.


Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Chloroplasts/enzymology , GTP Phosphohydrolases/metabolism , Intracellular Membranes/enzymology , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Alanine/metabolism , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/metabolism , Chloroplast Proteins , Circular Dichroism , Escherichia coli , Hydrolysis , Light-Harvesting Protein Complexes/metabolism , Lipid Bilayers/metabolism , Liposomes/metabolism , Molecular Sequence Data , Nucleotides/metabolism , Pisum sativum , Peptides/chemistry , Peptides/metabolism , Phenylalanine/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/metabolism , Structure-Activity Relationship , Thylakoids/metabolism
6.
Biomol NMR Assign ; 2(1): 37-39, 2008 Jun.
Article in English | MEDLINE | ID: mdl-19112521

ABSTRACT

We report the assignment of a 109 amino acid C-terminal chromo domain of the chloroplast signal recognition particle cpSRP43 subunit. cpSRP43 plays a crucial role in the targeting of light harvesting chlorophyll proteins to the thylakoids.

7.
J Mol Biol ; 381(1): 49-60, 2008 Aug 01.
Article in English | MEDLINE | ID: mdl-18586266

ABSTRACT

Signal recognition particle in chloroplasts (cpSRP) exhibits the unusual ability to bind and target full-length proteins to the thylakoid membrane. Unlike cytosolic SRPs in prokaryotes and eukaryotes, cpSRP lacks an RNA moiety and functions as a heterodimer composed of a conserved 54-kDa guanosine triphosphatase (cpSRP54) and a unique 43-kDa subunit (cpSRP43). Assembly of the cpSRP heterodimer is a prerequisite for post-translational targeting activities and takes place through interactions between chromatin modifier domain 2 (CD2) of cpSRP43 and a unique 10-amino-acid region in cpSRP54 (cpSRP54(pep)). We have used multidimensional NMR spectroscopy and other biophysical methods to examine the assembly and structure of the cpSRP43-cpSRP54 interface. Our data show that CD2 of cpSRP43 binds to cpSRP54(pep) in a 1:1 stoichiometry with an apparent K(d) of approximately 1.06 muM. Steady-state fluorescence and far-UV circular dichroism data suggest that the CD2-cpSRP54(pep) interaction causes significant conformational changes in both CD2 and the peptide. Comparison of the three-dimensional solution structures of CD2 alone and in complex with cpSRP54(pep) shows that significant structural changes are induced in CD2 in order to establish a binding interface contributed mostly by residues in the N-terminal segment of CD2 (Phe5-Val10) and an arginine doublet (Arg536 and Arg537) in the cpSRP54 peptide. Taken together, our results provide new insights into the mechanism of cpSRP assembly and the structural forces that stabilize the functionally critical cpSRP43-cpSRP54 interaction.


Subject(s)
Chloroplasts/metabolism , Signal Recognition Particle/chemistry , Signal Recognition Particle/metabolism , Amino Acid Sequence , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis/metabolism , Chloroplast Proteins , Chromatin/metabolism , Dimerization , Intracellular Membranes/metabolism , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Folding , Protein Structure, Tertiary , Signal Recognition Particle/genetics , Temperature , Titrimetry
8.
Biomol NMR Assign ; 2(1): 37-9, 2008 Jun.
Article in English | MEDLINE | ID: mdl-19636919

ABSTRACT

We report the assignment of a 109 amino acid C-terminal chromo domain of the chloroplast signal recognition particle cpSRP43 subunit. cpSRP43 plays a crucial role in the targeting of light harvesting chlorophyll proteins to the thylakoids.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Signal Recognition Particle/chemistry , Amino Acid Sequence , Carbon Isotopes/chemistry , Chloroplast Proteins , Molecular Sequence Data , Molecular Weight , Nitrogen Isotopes/chemistry , Protein Subunits , Protons
9.
Plant Cell ; 19(5): 1635-48, 2007 May.
Article in English | MEDLINE | ID: mdl-17513500

ABSTRACT

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) target proteins both cotranslationally and posttranslationally to the thylakoids. This dual function enables cpSRP to utilize its posttranslational activities for targeting a family of nucleus-encoded light-harvesting chlorophyll binding proteins (LHCPs), the most abundant membrane proteins in plants. Previous in vitro experiments indicated an absolute requirement for all cpSRP pathway soluble components. In agreement, a cpFtsY mutant in Arabidopsis thaliana exhibits a severe chlorotic phenotype resulting from a massive loss of LHCPs. Surprisingly, a double mutant, cpftsy cpsrp54, recovers to a great extent from the chlorotic cpftsy phenotype. This establishes that in plants, a new alternative pathway exists that can bypass cpSRP posttranslational targeting activities. Using a mutant form of cpSRP43 that is unable to assemble with cpSRP54, we complemented the cpSRP43-deficient mutant and found that this subunit is required for the alternative pathway. Along with the ability of cpSRP43 alone to bind the ALBINO3 translocase required for LHCP integration, our results indicate that cpSRP43 has developed features to function independently of cpSRP54/cpFtsY in targeting LHCPs to the thylakoid membranes.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Chloroplasts/metabolism , Signal Recognition Particle/metabolism , Arabidopsis/ultrastructure , Chlorophyll/metabolism , Chloroplast Proteins , Chloroplasts/ultrastructure , Dimerization , Fluorescence , Genetic Complementation Test , Light-Harvesting Protein Complexes/metabolism , Membrane Proteins/metabolism , Models, Biological , Mutation/genetics , Phenotype , Protein Binding , Protein Transport , Thylakoids/metabolism
10.
J Pharmacol Exp Ther ; 322(1): 30-9, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17452421

ABSTRACT

When generating monoclonal antibodies (mAb) against small molecules, the chemical composition and molecular orientation of the drug-like hapten on the antigen is a crucial determinant. This is especially important when attempting to discover therapeutic mAb against the drugs of abuse (+)-methamphetamine [(+)-METH], (+)-amphetamine [(+)-AMP], and the related compound (+)-3,4-methylenedioxymethamphetamine [(+)-MDMA, the plus isomer in the racemic mixture known as MDMA or ecstasy]. The goal of these studies was to design and synthesize (+)-METH-like haptens with structural attributes that could make them effective for generating monoclonal antibodies for treating medical problems associated with these stimulant drugs of abuse. Five prototype (+)-METH-like haptens, which mimic structural aspects of these drugs, were synthesized and used to generate mAb. After screening for anti-(+)-METH IgG antibodies in more than 25,000 potential mouse hybridoma cell lines, one prototype mAb from each of the five haptens was selected and studied in detail for molecular properties and preclinical efficacy. The amino acid sequences of the IgG-variable regions, structural models, affinity, and ligand specificity of each mAb were then used to help elucidate important therapeutic characteristics. Four of these antibodies exhibited high affinity and specificity to (+)-METH and (+)-MDMA; whereas one antibody (designated mAb4G9) exhibited high affinity and specificity to (+)-METH, (+)-MDMA, and (+)-AMP, without significant cross-reactivity against other METH-like ligands, over-the-counter medications, or endogenous neurotransmitters. Considered together, discovery of mAb4G9 and the other antibodies in this report represent an important step in understanding the process for custom design of drug class-specific therapeutic antibodies for the treatment of drug addiction.


Subject(s)
Amphetamine-Related Disorders/drug therapy , Antibodies, Monoclonal/therapeutic use , Drug Design , Haptens , Methamphetamine/toxicity , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Complementarity Determining Regions/chemistry , Cross Reactions , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Sequence Data , Structure-Activity Relationship
11.
J Biol Chem ; 280(50): 41465-71, 2005 Dec 16.
Article in English | MEDLINE | ID: mdl-16183644

ABSTRACT

Chloroplasts contain a unique signal recognition particle (cpSRP). Unlike the cytoplasmic forms, the cpSRP lacks RNA but contains a conserved 54-kDa GTPase and a novel 43-kDa subunit (cpSRP43). Recently, three functionally distinct chromodomains (CDs) have been identified in cpSRP43. In the present study, we report the three-dimensional solution structures of the three CDs (CD1, CD2, and CD3) using a variety of triple resonance NMR experiments. The structure of CD1 consists of a triple-stranded beta-sheet segment. The C-terminal helical segment typically found in the nuclear chromodomains is absent in CD1. The secondary structural elements in CD2 and CD3 include a triple-stranded antiparallel beta-sheet and a C-terminal helix. Interestingly, the orientation of the C-terminal helix is significantly different in the structures of CD2 and CD3. Critical comparison of the structures of the chromodomains of cpSRP43 with those found in nuclear chromodomain proteins revealed that the diverse protein-protein interactions mediated by the CDs appear to stem from the differences that exist in the surface charge potentials of each CD. Results of isothermal titration calorimetry experiments confirmed that only CD2 is involved in binding to cpSRP54. The negatively charged C-terminal helix in CD2 possibly plays a crucial role in the cpSRP54-cpSRP43 interaction.


Subject(s)
Chloroplasts/metabolism , Signal Recognition Particle/physiology , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Calorimetry , Chloroplast Proteins , Cytoplasm/metabolism , Escherichia coli/metabolism , Light-Harvesting Protein Complexes , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Photosynthetic Reaction Center Complex Proteins , Plant Proteins , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA/chemistry , Sequence Homology, Amino Acid , Signal Recognition Particle/chemistry
12.
Biotechnol Bioeng ; 88(1): 77-83, 2004 Oct 05.
Article in English | MEDLINE | ID: mdl-15389486

ABSTRACT

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 Specificity
13.
J Biol Chem ; 279(41): 43077-84, 2004 Oct 08.
Article in English | MEDLINE | ID: mdl-15292240

ABSTRACT

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 Techniques
14.
J Cell Biol ; 162(7): 1245-54, 2003 Sep 29.
Article in English | MEDLINE | ID: mdl-14517205

ABSTRACT

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 Specificity
15.
J Gen Physiol ; 121(5): 477-93, 2003 May.
Article in English | MEDLINE | ID: mdl-12719487

ABSTRACT

The hydrophobic coupling between membrane-spanning proteins and the lipid bilayer core causes the bilayer thickness to vary locally as proteins and other "defects" are embedded in the bilayer. These bilayer deformations incur an energetic cost that, in principle, could couple membrane proteins to each other, causing them to associate in the plane of the membrane and thereby coupling them functionally. We demonstrate the existence of such bilayer-mediated coupling at the single-molecule level using single-barreled as well as double-barreled gramicidin channels in which two gramicidin subunits are covalently linked by a water-soluble, flexible linker. When a covalently attached pair of gramicidin subunits associates with a second attached pair to form a double-barreled channel, the lifetime of both channels in the assembly increases from hundreds of milliseconds to a hundred seconds--and the conductance of each channel in the side-by-side pair is almost 10% higher than the conductance of the corresponding single-barreled channels. The double-barreled channels are stabilized some 100,000-fold relative to their single-barreled counterparts. This stabilization arises from: first, the local increase in monomer concentration around a single-barreled channel formed by two covalently linked gramicidins, which increases the rate of double-barreled channel formation; and second, from the increased lifetime of the double-barreled channels. The latter result suggests that the two barrels of the construct associate laterally. The underlying cause for this lateral association most likely is the bilayer deformation energy associated with channel formation. More generally, the results suggest that the mechanical properties of the host bilayer may cause the kinetics of membrane protein conformational transitions to depend on the conformational states of the neighboring proteins.


Subject(s)
Gramicidin/chemistry , Ion Channels/chemistry , Lipid Bilayers/chemistry , Circular Dichroism , Dimerization , Electric Conductivity , Hydrophobic and Hydrophilic Interactions , In Vitro Techniques , Models, Biological , Models, Molecular , Permeability , Protein Conformation , Thermodynamics
16.
J Biol Chem ; 277(35): 32400-4, 2002 Aug 30.
Article in English | MEDLINE | ID: mdl-12105232

ABSTRACT

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/metabolism
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