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Results for “Protein Structure, Quaternary”

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PubMed2002

Secondary and quaternary structures of the (+)-pinoresinol-forming dirigent protein.

The (+)-pinoresinol-forming dirigent protein is the first protein capable of stereoselectively coupling two coniferyl alcohol derived radical species, in this case to give the 8-8' linked (+)-pinoresinol. Only dimeric cross-linked dirigent protein structures were isolated when 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide was used as cross-linking agent, whereas the associated oxidase, presumed to generate the corresponding free radical substrate, was not detected. Native Forsythia intermedia dirigent protein isoforms were additionally subjected to MALDI-TOF and ESI-MS analyses, which established the presence of both monomeric masses of 23-25 kDa and dimeric dirigent protein species ranging from 46 to 49 kDa. Analytical ultracentrifugation, sedimentation velocity, and sedimentation equilibrium analyses of the native dirigent protein in open solution confirmed further its dimeric nature as well as a propensity to aggregate, with the latter being dependent upon both temperature and solution ionic strength. Circular dichroism analysis suggested that the dirigent protein was primarily composed of beta-sheet and loop structures.

Chromatography, Gel↗

PubMed2006

Domain organization and quaternary structure of the Saccharomyces cerevisiae silent information regulator 3 protein, Sir3p.

The silent information regulator protein 3 (Sir3p) functions in the initiation, propagation, and maintenance of transcriptionally silenced chromatin in Saccharomyces cerevisiae. To better understand the physicochemical basis for its effects on chromatin architecture, recombinant full-length S. cerevisiae Sir3p has been purified to near homogeneity on the large-scale and characterized by circular dichroism, limited protease digestion, and analytical ultracentrifugation. Results indicate that the Sir3p monomer has a unique tripartite domain organization, including a nearly 300-amino-acid residue stretch of intrinsically disordered residues that lies internal to its structured N- and C-terminal regions. Sir3p self-associates extensively in moderate salt and at micromolar protein concentrations producing a broad range of oligomers that sediment from 8 to in excess of 85 S. These results provide new insight into Sir3p domain organization and quaternary structure and support a nucleosome bridging model for Sir3p-dependent regulation of chromatin architecture.

Animals↗

PubMed2002

Core mutations switch monomeric protein GB1 into an intertwined tetramer.

The structure of a mutant immunoglobulin-binding B1 domain of streptococcal protein G (GB1), which comprises five conservative changes in hydrophobic core residues, was determined by NMR spectroscopy and X-ray crystallography. The oligomeric state and quaternary structure of the mutant protein are drastically changed from the wild type protein. The mutant structure consists of a symmetric tetramer, with intermolecular strand exchange involving all four units. Four of the five secondary structure elements present in the monomeric wild type GB1 structure are retained in the tetrameric structure, although their intra- and intermolecular interactions are altered. Our results demonstrate that through the acquisition of a moderate number of pivotal point mutations, proteins such as GB1 are able to undergo drastic structural changes, overcoming reduced stability of the monomeric unit by multimerization. The present structure is an illustrative example of how proteins exploit the breadth of conformational space.

Amino Acid Sequence↗

PubMed2005

The Cockayne syndrome group B protein is a functional dimer.

Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by developmental abnormalities, UV sensitivity, and premature aging. The CS group B (CSB) protein belongs to the SNF2-family of DNA-dependent ATPases and is implicated in transcription elongation, transcription coupled repair, and base excision repair. It is a DNA stimulated ATPase and remodels chromatin in vitro. We demonstrate for the first time that full-length CSB positively cooperates in ATP hydrolysis as a function of protein concentration. We have investigated the quaternary structure of CSB using a combination of protein-protein complex trapping experiments and gel filtration, and found that CSB forms a dimer in solution. Chromatography studies revealed that enzymatically active CSB has an apparent molecular mass of approximately 360 kDa, consistent with dimerization of CSB. Importantly, in vivo protein cross-linking showed the presence of the CSB dimer in the nucleus of HeLa cells. We further show that dimerization occurs through the central ATPase domain of the protein. These results have implications for the mechanism of action of CSB, and suggest that other SNF2-family members might also function as dimers.

Adenosine Triphosphatases↗

PubMedDate not supplied

Investigation of the thermal stability of porin from Paracoccus denitrificans by site-directed mutagenesis and Fourier transform infrared spectroscopy.

The folding of membrane proteins was addressed using outer membrane protein porin from the soil bacterium Paracoccus denitrificans (P. den.). IR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were used to probe the effect of mutagenesis on the thermal stability of the protein. Secondary structure analysis by amide I ir spectroscopy showed that the wild-type protein was predominantly composed of beta-sheet, which supports the x-ray crystal structure information (A. Hirsch, J. Breed, K. Saxena, O.-M. H. Richter, B. Ludwig, K. Diederichs, and W. Welte, FEBS Letters, 1997, Vol. 404, pp. 208-210). The mutants E81Q, W74C, and E81Q/D148N were shown to have similar secondary structure composition as the wild type. Wild-type protein and the mutants in detergent micelles underwent irreversible denaturation as a result of heating. Transition temperature calculated from the amide I analysis revealed that mutant porins were slightly less stable compared to the wild type. The protein in micelles showed complete monomerization of the trimer above 85 degrees C. In native-like conditions (provided by liposomes), no change was observed in the secondary structure of the protein until 95 degrees C. This is supported by SDS-PAGE as no change in quaternary structure was observed, proving that the proteins are structurally thermostable in liposomes as compared to micelles. Our studies demonstrated that porins resistant to detergents and proteases are highly thermostable as well.

Cell Membrane↗

PubMed2005

Crystallization and preliminary X-ray diffraction analysis of thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1.

Thioredoxin peroxidase is a member of the peroxiredoxin family and plays a dominant role in a hydrogen peroxide metabolism. A recombinant form of the hyperthermostable thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1, a polypeptide consisting of 250 amino acids, was purified. The C207S mutant protein was crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as the precipitant at 298 K. Diffraction data were collected and processed to 2.7 A resolution. The crystal belongs to space group P1, with unit-cell parameters a = 126.2, b = 126.3, c = 213.7 A, alpha = 80.4, beta = 80.3, gamma = 70.7 degrees. Calculation of the self-rotation function showed that the protein quaternary structure includes a fivefold axis and five twofold axes.

Aeropyrum↗

PubMed2001

Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of conserved glycine residues and the identification of additional conserved protein motifs in eNTPDases.

Glycine residues are recognized as important structural determinants in nucleotide-binding domains of many enzymes. The functional significance of seven glycine residues invariant in all 22 eNTPDase sequences was therefore examined. Glycine-to-alanine mutants of eNTPDase3 were analyzed for nucleotidase activities and tertiary and quaternary structure changes. Mutations G98A and G183A had modest effects on ATPase and ADPase activities. The G141A mutation resulted in 4- to 5-fold decreased nucleotidase activity, while the G222A mutation decreased ATPase activity 20-fold, and ADPase activity 6-fold. Unlike the other five glycine mutants, the G263A and G462A mutations caused significant loss of nucleotidase activity which was observed concomitant with lower protein expression levels, large-scale changes in tertiary and quaternary protein structure, and decreased trafficking to the plasma membrane. Thus, these data identify glycine residues that are essential for enzymatic activity and the tertiary and quaternary structure of eNTPDase3. Further, two additional conserved regions in the eNTPDases are identified, apyrase conserved regions ACR1a and ACR4a, which may be involved in phosphate binding/hydrolysis and protein folding, respectively.

Adenosine Triphosphatases↗

PubMed2001

The effect of water on the rate of conformational change in protein allostery.

The influence of solvation on the rate of quaternary structural change is investigated in human hemoglobin, an allosteric protein in which reduced water activity destabilizes the R state relative to T. Nanosecond absorption spectroscopy of the heme Soret band was used to monitor protein relaxation after photodissociation of aqueous HbCO complex under osmotic stress induced by the nonbinding cosolute poly(ethylene glycol) (PEG). Photolysis data were analyzed globally for six exponential time constants and amplitudes as a function of osmotic stress and viscosity. Increases in time constants associated with geminate rebinding, tertiary relaxation, and quaternary relaxation were observed in the presence of PEG, along with a decrease in the fraction of hemes rebinding CO with the slow rate constant characteristic of the T state. An analysis of these results along with those obtained by others for small cosolutes showed that both osmotic stress and solvent viscosity are important determinants of the microscopic R --> T rate constant. The size and direction of the osmotic stress effect suggests that at least nine additional water molecules are required to solvate the allosteric transition state relative to the R-state hydration, implying that the transition state has a greater solvent-exposed area than either end state.

Allosteric Regulation↗

PubMed2006

The cataract-causing mutation G98R in human alphaA-crystallin leads to folding defects and loss of chaperone activity.

PURPOSE: The objective of this study is to understand the molecular basis of cataract that develops due to the mutation of the glycine-98 residue to arginine in alphaA-crystallin. METHODS: The glycine-98 residue was mutated to arginine by site-directed mutagenesis. The expression, structural and chaperone properties and thermal stability of the mutant, G98RalphaA-crystallin have been studied. The secondary and tertiary structure of the wild type and the mutant protein was studied using circular dichroism and fluorescence spectroscopy. The quaternary structure was studied by gel filtration chromatography and dynamic light scattering. Chaperone activity studies were carried out using DTT-induced aggregation of insulin. RESULTS: Unlike the wild type protein, the heterologous expression of G98R alphaA-crystallin in E. coli results in the formation of inclusion bodies. Upon dissolving the inclusion bodies in 3 M urea and subjecting to refolding, it yielded a clear solution. The refolded mutant protein exhibits altered secondary, tertiary and quaternary structure, which lacks chaperone function, and is susceptible to heat-induced aggregation. CONCLUSIONS: The G98R mutation in alphaA-crystallin results in altered folding and becomes aggregation-prone leading to formation of large oligomers lacking chaperone function. Tendency to aggregate and loss of chaperone activity could be contributing to turbidity and loss of transparency of the lens.

Arginine↗

PubMed2005

pH-dependent substrate preference of pig heart lipoamide dehydrogenase varies with oligomeric state: response to mitochondrial matrix acidification.

Cycling of intracellular pH has recently been shown to play a critical role in ischemia-reperfusion injury. Ischemia-reperfusion also leads to mitochondrial matrix acidification and dysfunction. However, the mechanism by which matrix acidification contributes to mitochondrial dysfunction, oxidative stress, and the resultant cellular injury has not been elucidated. We observe pH-dependent equilibria between monomeric, dimeric, and a previously undescribed tetrameric form of pig heart lipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme. Dynamic light scattering studies of native LADH in aqueous solution indicate that lowering pH favors a shift in average molecular mass from higher oligomeric states to monomer. Sedimentation velocity of LADH entrapped in reverse micelles reveals dimer and tetramer at both pH 5.8 and 7.5, but monomer was observed only at pH 5.8. Enzyme activity measurements in reverse Aerosol OT micelles in octane indicate that LADH dimer and tetramer possess lipoamide dehydrogenase and diaphorase activities at pH 7.5. Upon acidification to pH 5.8 only the LADH monomer is active and only the diaphorase activity is observed. These results indicate a correlation between pH-dependent changes in the LADH reaction specificity and its oligomeric state. The acidification of mitochondrial matrix that occurs during ischemia-reperfusion injury is sufficient to alter the structure and enzymatic specificity of LADH, thereby reducing mitochondrial defenses, increasing oxidative stress, and slowing the recovery of energy metabolism. Matrix acidification may also disrupt the quaternary structure of other mitochondrial protein complexes critical for cellular homeostasis and survival.

2,6-Dichloroindophenol↗

PubMed2000

Native quaternary structure of bovine alpha-crystallin.

Alpha-crystallin is the most important soluble protein in the eye lens. It is responsible for creating a high refractive index and is known to be a small heat-shock protein. We have used static and dynamic light scattering to study its quaternary structure as a function of isolation conditions, temperature, time, and concentration. We have used tryptophan fluorescence to study the temperature dependence of the tertiary structure and its reversibility. Gel filtration, analytical ultracentrifugation, polyacrylamide gel electrophoretic analysis, and absorption measurements were used to study the chaperone-like activity of alpha-crystallin in the presence of destabilized lysozyme. We have demonstrated that the molecular mass of the in vivo alpha-crystallin oligomer is about 700 kDa (alpha(native)) while the 550 kDa molecule (alpha(37 degrees C),diluted), which is often found in vitro, is a product of prolonged storage at 37 degrees C of low concentrated alpha-crystallin solutions. We have proven that the molecular mass of the alpha-crystallin oligomer is concentration dependent at 37 degrees C. We have found strong indications that, during chaperoning, the alpha-crystallin oligomer undergoes a drastic rearrangement of its peptides during the process of complex formation with destabilized lysozyme. We propose the hypothesis that all these processes are governed by the phenomenon of subunit exchange, which is well-known to be strongly temperature-dependent.

Animals↗

PubMed2004

Protein engineering of pyruvate carboxylase: investigation on the function of acetyl-CoA and the quaternary structure.

Pyruvate carboxylase (PC) from Bacillus thermodenitrificans was engineered in such a way that the polypeptide chain was divided into two, between the biotin carboxylase (BC) and carboxyl transferase (CT) domains. The two proteins thus formed, PC-(BC) and PC-(CT+BCCP), retained their catalytic activity as assayed by biotin-dependent ATPase and oxamate-dependent oxalacetate decarboxylation, for the former and the latter, respectively. Neither activity was dependent on acetyl-CoA, in sharp contrast to the complete reaction of intact PC. When assessed by gel filtration chromatography, PC-(BC) was found to exist either in dimers or monomers, depending on the protein concentration, while PC-(CT + BCCP) occurred in dimers for the most part. The two proteins do not associate spontaneously or in the presence of acetyl-CoA. Based on these observations, this paper discusses how the tetrameric structure of PC is built up and how acetyl-CoA modulates the protein structure.

Acetyl Coenzyme A↗

PubMed2003

Immunodominance of conformation-dependent B-cell epitopes of protein antigens.

Immunodominance of conformational epitopes over linear ones in four proteins was quantified making use of the B-cell hybridoma technology. The proteins were immunized in their native forms into BALB/c mice, and clonal frequencies of B-cell hybridomas that produce antibodies to the native and denatured forms were determined, using ELISA and immunoblotting. All 16 monoclonal antibodies (mAbs) to Porphyromonas gingivalis fimbria were suggested to recognize conformational epitopes expressed by the oligomer. Ten out of 14 mAbs to Serratia marcescens fimbria and 13 of 15 mAbs to hen lysozyme were also specific to their conformational epitopes. In contrast, all 18 mAbs to a surface protein of Streptococcus mutans, termed PAc, reacted to both the native and denatured forms, thereby indicating the immunodominance of linear epitopes in this protein. The results suggest that B-cell epitopes of proteins possessing stable tertiary or quaternary structures are predominantly expressed by the higher-order structures.

Animals↗

PubMed2005

Structures of two superoxide dismutases from Bacillus anthracis reveal a novel active centre.

The BA4499 and BA5696 genes of Bacillus anthracis encode proteins homologous to manganese superoxide dismutase, suggesting that this organism has an expanded repertoire of antioxidant proteins. Differences in metal specificity and quaternary structure between the dismutases of prokaryotes and higher eukaryotes may be exploited in the development of therapeutic antibacterial compounds. Here, the crystal structure of two Mn superoxide dismutases from B. anthracis solved to high resolution are reported. Comparison of their structures reveals that a highly conserved residue near the active centre is substituted in one of the proteins and that this is a characteristic feature of superoxide dismutases from the B. cereus/B. anthracis/B. thuringiensis group of organisms.

Bacillus anthracis↗

PubMed2001

Weak protein-protein interactions in lectins: the crystal structure of a vegetative lectin from the legume Dolichos biflorus.

The legume lectins are widely used as a model system for studying protein-carbohydrate and protein-protein interactions. They exhibit a fascinating quaternary structure variation, which becomes important when they interact with multivalent glycoconjugates, for instance those on cell surfaces. Recently, it has become clear that certain lectins form weakly associated oligomers. This phenomenon may play a role in the regulation of receptor crosslinking and subsequent signal transduction. The crystal structure of DB58, a dimeric lectin from the legume Dolichos biflorus reveals a separate dimer of a previously unobserved type, in addition to a tetramer consisting of two such dimers. This tetramer resembles that formed by DBL, the seed lectin from the same plant. A single amino acid substitution in DB58 affects the conformation and flexibility of a loop in the canonical dimer interface. This disrupts the formation of a stable DBL-like tetramer in solution, but does not prohibit its formation in suitable conditions, which greatly increases the possibilities for the cross-linking of multivalent ligands. The non-canonical DB58 dimer has a buried symmetrical alpha helix, which can be present in the crystal in either of two antiparallel orientations. Two existing structures and datasets for lectins with similar quaternary structures were reconsidered. A central alpha helix could be observed in the soybean lectin, but not in the leucoagglutinating lectin from Phaseolus vulgaris. The relative position and orientation of the carbohydrate-binding sites in the DB58 dimer may affect its ability to crosslink mulitivalent ligands, compared to the other legume lectin dimers.

Amino Acid Sequence↗

PubMed1995

Calcium-induced dimerization of troponin C: mode of interaction and use of trifluoroethanol as a denaturant of quaternary structure.

Protein aggregation can be a problem, especially as a large number of proteins become available for structural studies at fairly high concentrations using solution techniques such as NMR spectroscopy. The muscle regulatory protein troponin C (TnC) undergoes a calcium-induced dimerization at neutral pH with a dissociation constant for the dimerization of 0.4 mM at 20 degrees C. The present study indicates that the mode of dimerization involves the N-domain of one monomer interacting with the N-domain of another monomer. Addition of the solvent trifluoroethanol (TFE) to a concentration of 15%, v/v, results in a 10-fold increase in the dimer dissociation constant of calcium-saturated TnC (4 mM at 20 degrees C), making TnC predominantly a monomer for spectroscopic studies. Further, TFE, at the concentrations used herein, acts to perturb the quaternary structure of TnC without adversely affecting the secondary or tertiary structure as evidenced by minimal changes to its CD spectra and 1H, 13C, and 15N NMR chemical shifts.

Animals↗

PubMed2006

Inhibition of Escherichia coli heat-labile enterotoxin B subunit pentamer (EtxB5) assembly in vitro using monoclonal antibodies.

Heat-labile enterotoxin (Etx) produced by certain strains of Escherichia coli is a major virulence factor related to cholera toxin. Both are hexameric proteins comprising one A-subunit and five B-subunits. The pentameric B-subunit of E. coli has a high affinity for G(M1)-ganglioside receptors on gut epithelial cells and is directly responsible for toxin entry. The pentameric B-subunit (EtxB(5)) is an exceptionally stable protein, being able to maintain its quaternary structure over a wide pH range (2.0- 11.0). However, little is known about the formation of the pentameric structure (EtxB(5)) from newly synthesized B-subunit monomers (EtxB(1)). We previously described and characterized a mAb (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB(1) (Amin, T., Larkins, A., James, R. F. L., and Hirst, T. R. (1995) J. Biol. Chem. 270, 20143-20150). Here we also describe a mAb (LDS16) with exquisite specificity for pentameric EtxB. In this study, we have used these two mAbs, in combination, to probe the in vitro assembly of EtxB(5) from EtxB(1). EtxB pentamers disassemble in highly acidic conditions, giving rise to monomeric B-subunits that can reassemble if placed in buffers of neutral pH. Using this in vitro assembly model, it was found that at a molar ratio of 1:1; LDS47:EtxB, 50% of reassembly was inhibited, and that this inhibition increased to 90% at a ratio of 2:1. These results infer that the N-terminal decapeptide region (APQSITELCS) defined by the LDS47 antibody is crucial for competent pentameric B-subunit assembly and stabilization.

Animals↗

PubMed2004

Modulation of the Leishmania donovani peroxin 5 quaternary structure by peroxisomal targeting signal 1 ligands.

The import of proteins containing the peroxisomal targeting signal 1 (PTS1) into the Leishmania glycosome is dependent on the docking of the PTS1-loaded LdPEX5 cytosolic receptor with LdPEX14 on the glycosome surface. Here we show that, in the absence of PTS1, LdPEX5 is a tetramer that is stabilized by two distinct interaction domains; the first is a coiled-coil motif encompassing residues 277 to 310, whereas the second domain is localized to residues 1 to 202. By using microcalorimetry, surface plasmon resonance, and size exclusion chromatography techniques, we show that PTS1 peptide binding to LdPEX5 tetramers promotes their dissociation into dimeric structures, which are stabilized by a coiled-coil interaction. Moreover, we demonstrated that the resulting LdPEX5-PTS1 complex is remarkably stable and exhibits extremely slow dissociation kinetics. However, binding of LdPEX14 to LdPEX5 modulates the LdPEX5-PTS1 affinity as it decreases the thermodynamic dissociation constant for this latter complex by 10-fold. These changes in the oligomeric state of LdPEX5 and in its affinity for PTS1 ligand upon LdPEX14 binding may explain how, under physiological conditions, LdPEX5 can function to deliver and unload its cargo to the protein translocation machinery on the glycosomal membrane.

Amino Acid Sequence↗