Co-chaperonin GroES subunit exchange as dependent on time, pH, protein concentration, and urea.

The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES7) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES7 and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES7) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel. For each sample, there are eight Coomassie-stained electrophoretic bands showing different charges that result from a different number of included mutant subunits, each carrying an additional negative charge. The intensities of these bands serve to analyze the protein subunit exchange. The protein stability is evaluated using the transverse urea gradient gel electrophoresis (TUGGE). At pH 8.0, the intensities of the initial bands corresponding to WT-GroES7 and CMC-GroES7 are decreased with a half-time of (23 ±â€¯2) min. The exchange decreases with decreasing pH and seems to be strongly hindered at pH 5.2 due to the protonation of groups with pK ~ 6.3, which stabilizes the protein quaternary structure. The destabilization of the protein quaternary structure caused by increased pH, decreased protein concentration, or urea accelerates the GroES subunit exchange. This study allows visualizing the subunit exchange in oligomeric proteins and confirms its direct connection with the stability of the protein quaternary structure.

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties.

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

Recombinant Globular Domain of TcpA Pilin from Vibrio cholerae El Tor: Recovery from Inclusion Bodies and Structural Characterization.

The production of recombinant proteins in Escherichia coli cells is often hampered by aggregation of newly synthesized proteins and formation of inclusion bodies. Here we propose the use of transverse urea gradient electrophoresis (TUGE) in testing the capability of folding of a recombinant protein from inclusion bodies dissolved in urea. A plasmid encoding the amino acid sequence 55-224 of TcpA pilin (C-terminal globular domain: TcpA-C) from Vibrio cholerae El Tor enlarged by a His-tag on its N-terminus was expressed in E. coli cells. The major fraction (about 90%) of the target polypeptide was detected in cell debris. The polypeptide was isolated from the soluble fraction and recovered from inclusion bodies after their urea treatment. Some structural properties of the polypeptide from each sample proved identical. The refolding protocol was developed on the basis of TUGE data and successfully used for the protein large-scale recovery from inclusion bodies. Spectral, hydrodynamic, and thermodynamic characteristics of the recombinant TcpA recovered from inclusion bodies indicate the presence of a globular conformation with a pronounced secondary structure and a rigid tertiary structure, which is promising for the design of immunodiagnostics preparations aimed to assess the pilin level in different strains of V. cholerae and to develop cholera vaccines.

The Denaturant- and Mutation-Induced Disassembly of Pseudomonas aeruginosa Hexameric Hfq Y55W Mutant.

Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced disassembly of the hexameric mutant Y55W of the Qß host factor (Hfq) from mesophilic Pseudomonas aeruginosa (Pae). Using intrinsic tryptophan fluorescence, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC), we show that the dissociation of Hfq Y55W occurs either under the effect of GuHCl or during the pre-denaturing transition, when the protein concentration is decreased, with both events proceeding through the accumulation of stable intermediate states. With an extremely low pH of 1.4, a low ionic strength, and decreasing protein concentration, the accumulated trimers and dimers turn into monomers. Also, we report on the structural features of monomeric Hfq resulting from a triple mutation (D9A/V43R/Y55W) within the inter-subunit surface of the protein. This globular and rigidly packed monomer displays a high thermostability and an oligomer-like content of the secondary structure, although its urea resistance is much lower.

Back to GroEL-Assisted Protein Folding: GroES Binding-Induced Displacement of Denatured Proteins from GroEL to Bulk Solution.

The main events in chaperone-assisted protein folding are the binding and ligand-induced release of substrate proteins. Here, we studied the location of denatured proteins previously bound to the GroEL chaperonin resulting from the action of the GroES co-chaperonin in the presence of Mg-ATP. Fluorescein-labeled denatured proteins (α-lactalbumin, lysozyme, serum albumin, and pepsin in the presence of thiol reagents at neutral pH, as well as an early refolding intermediate of malate dehydrogenase) were used to reveal the effect of GroES on their interaction with GroEL. Native electrophoresis has demonstrated that these proteins tend to be released from the GroEL-GroES complex. With the use of biotin- and fluorescein-labeled denatured proteins and streptavidin fused with luciferase aequorin (the so-called streptavidin trap), the presence of denatured proteins in bulk solution after GroES and Mg-ATP addition has been confirmed. The time of GroES-induced dissociation of a denatured protein from the GroEL surface was estimated using the stopped-flow technique and found to be much shorter than the proposed time of the GroEL ATPase cycle.

Chaperonin GroEL reassembly: an effect of protein ligands and solvent composition.

Chaperonin GroEL is a complex oligomeric heat shock protein (Hsp60) assisting the correct folding and assembly of other proteins in the cell. An intriguing question is how GroEL folds itself. According to the literature, GroEL reassembly is dependent on chaperonin ligands and solvent composition. Here we demonstrate dependence of GroEL reassembly efficiency on concentrations of the essential factors (Mg2+, ADP, ATP, GroES, ammonium sulfate, NaCl and glycerol). Besides, kinetics of GroEL oligomerization in various conditions was monitored by the light scattering technique and proved to be two-exponential, which suggested accumulation of a certain oligomeric intermediate. This intermediate was resolved as a heptamer by nondenaturing blue electrophoresis of GroEL monomers during their assembly in the presence of both Mg-ATP and co-chaperonin GroES. Presumably, this intermediate heptamer plays a key role in formation of the GroEL tetradecameric particle. The role of co-chaperonin GroES (Hsp10) in GroEL assembly is also discussed.

Amyloidogenic peptides of yeast cell wall glucantransferase Bgl2p as a model for the investigation of its pH-dependent fibril formation.

The pH-dependence of the ability of Bgl2p to form fibrils was studied using synthetic peptides with potential amyloidogenic determinants (PADs) predicted in the Bgl2p sequence. Three PADs, FTIFVGV, SWNVLVA and NAFS, were selected on the basis of combination of computational algorithms. Peptides AEGFTIFVGV, VDSWNVLVAG and VMANAFSYWQ, containing these PADs, were synthesized. It was demonstrated that these peptides had an ability to fibrillate at pH values from 3.2 to 5.0. The PAD-containing peptides, except for VDSWNVLVAG, could fibrillate also at pH values from pH 5.0 to 7.6. We supposed that the ability of Bgl2p to form fibrils most likely depended on the coordination of fibrillation activity of the PAD-containing areas and Bgl2p could fibrillate at mild acid and neutral pH values and lose the ability to fibrillate with the increasing of pH values. It was demonstrated that Bgl2p was able to fibrillate at pH value 5.0, to form fibrils of various morphology at neutral pH values and lost the fibrillation ability at pH value 7.6. The results obtained allowed us to suggest a new simple approach for the isolation of Bgl2p from Saccharomyces cerevisiae cell wall.

The major mRNP protein YB-1: structural and association properties in solution.

YB-1 is a major mRNP protein participating in the regulation of transcription and translation of a wide range of eukaryotic genes in many organisms probably due to its influence on mRNA packing into mRNPs. While the functional properties of YB-1 are extensively studied, little is known about its structural properties. In the present work we focused on studying its secondary structure, rigidity of its tertiary structure, compactness, and oligomerization in vitro by using far UV-CD, DSC, one-dimensional (1)H NMR, SAXS, sedimentation and FPLC. It was shown that only the cold shock domain within the entire YB-1 chain has a well-packed tertiary structure undergoing cooperative heat and cold denaturation transitions. In contrast, the rest of the YB-1 molecule is not rigidly packed and consists of PP II-like helical secondary structure elements and coil-like regions. At the same time, the overall dimension of the protein molecule is unexpectedly small. The polypeptide chains of YB-1 have a high tendency to form oligomers at neutral pH, while the extent and structural organization of the oligomers depend on protein concentration and ionic strength varying from compact monomeric units up to high molecular weight oligomers. These oligomers in solution are unstable and dissociate upon protein concentration decrease.

Folding intermediate and folding nucleus for I-->N and U-->I-->N transitions in apomyoglobin: contributions by conserved and nonconserved residues.

Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the phi-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I-->N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I-->N transition.

GroEL-assisted protein folding: does it occur within the chaperonin inner cavity?

The folding of protein molecules in the GroEL inner cavity under the co-chaperonin GroES lid is widely accepted as a crucial event of GroEL-assisted protein folding. This review is focused on the data showing that GroEL-assisted protein folding may proceed out of the complex with the chaperonin. The models of GroEL-assisted protein folding assuming ligand-controlled dissociation of nonnative proteins from the GroEL surface and their folding in the bulk solution are also discussed.

Symmetrical refolding of protein domains and subunits: example of the dimeric two-domain 3-isopropylmalate dehydrogenases.

The refolding mechanism of the homodimeric two-domain 3-isopropylmalate dehydrogenase (IPMDH) from the organisms adapted to different temperatures, Thermus thermophilus (Tt), Escherichia coli (Ec), and Vibrio sp. I5 (Vib), is described. In all three cases, instead of a self-template mechanism, the high extent of symmetry and cooperativity in folding of subunits and domains have been concluded from the following experimental findings: The complex time course of refolding, monitored by Trp fluorescence, consists of a fast (the rate constant varies as 16.5, 25.0, and 11.7 min-1 in the order of Tt, Ec, and Vib IPMDHs) and a slow (the rate constants are 0.11, 0.80, and 0.23 min-1 for the three different species) first-order process. However, a burst increase of Trp fluorescence anisotropy to the value of the native states indicates that in all three cases the association of the two polypeptide chains occurs at the beginning of refolding. This dimeric species binds the substrate IPM, but the native-like interactions of the tertiary and quaternary structures are only formed during the slow phase of refolding, accompanied by further increase of protein fluorescence and appearance of FRET between Trp side chain(s) and the bound NADH. Joining the contacting arms of each subunit also takes place exclusively during this slow phase. To monitor refolding of each domain within the intact molecule of T. thermophilus IPMDH, Trp's (located in separate domains) were systematically replaced with Phe's. The refolding processes of the mutants were followed by measuring changes in Trp fluorescence and in FRET between the particular Trp and NADH. The high similarity of time courses (both in biphasicity and in their rates) strongly suggests cooperative folding of the domains during formation of the native three-dimensional structure of IPMDH.

Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli.

PsbO protein is an important constituent of the water-oxidizing complex, located on the lumenal side of photosystem II. We report here the efficient expression of the spinach PsbO in E. coli where the solubility depends entirely on the formation of the disulfide bond. The PsbO protein purified from a pET32 system that includes thioredoxin fusion is properly folded and functionally active. Urea unfolding experiments imply that the reduction of the single disulfide bridge decreases stability of the protein. Analysis of inter-residue contact density through the PsbO molecule shows that Cys51 is located in a cluster with high contact density. Reduction of the Cys28-Cys51 bond is proposed to perturb the packing interactions in this cluster and destabilize the protein as a whole. Taken together, our results give evidence that PsbO exists in solution as a compact highly ordered structure, provided that the disulfide bridge is not reduced.

Rates of unfolding, rather than refolding, determine thermal stabilities of thermophilic, mesophilic, and psychrotrophic 3-isopropylmalate dehydrogenases.

The relationship between the thermal stability of proteins and rates of unfolding and refolding is still an open issue. The data are very scarce, especially for proteins with complex structure. Here, time-dependent denaturation-renaturation experiments on Thermus thermophilus, Escherichia coli, and Vibrio sp. I5 3-isopropylmalate dehydrogenases (IPMDHs) of different heat stabilities are presented. Unfolding, as monitored by several methods, occurs in a single first-order step with half-times of approximately 1 h, several minutes, and few seconds for the thermophilic, mesophilic, and psychrotrophic enzymes, respectively. The binding of Mn*IPM (the manganese complex of 3-isopropylmalate) markedly reduces the rates of unfolding; this effect is more prominent for the less stable enzyme variants. Refolding is a two-step or multistep first-order process involving an inactive intermediate(s). The restoration of the native structure and reactivation take place with a half-time of a few minutes for all three IPMDHs. Thus, the comparative experimental unfolding-refolding studies of the three IPMDHs with different thermostabilities have revealed a close relationship between thermostability and unfolding rate. Structural analysis has shown that the differences in the molecular contacts between selected nonconserved residues are responsible for the different rates of unfolding. On the other hand, the folding rates might be correlated with the absolute contact order, which does not significantly vary between IPMDHs with different thermostabilities. On the basis of our observations, folding rates appear to be dictated by global structural characteristics (such as native topology, i.e., contact order) rather than by thermodynamic stability.

Three-state protein folding: experimental determination of free-energy profile.

When considering protein folding with a transient intermediate, a difficulty arises as to determination of the rates of separate transitions. Here we overcome this problem, using the kinetic studies of the unfolding/refolding reactions of the three-state protein apomyoglobin as a model. Amplitudes of the protein refolding kinetic burst phase corresponding to the transition from the unfolded (U) to intermediate (I) state, that occurs prior to the native state (N) formation, allow us to estimate relative populations of the rapidly converting states at various final urea concentrations. On the basis of these proportions, a complicated experimental chevron plot has been deconvolved into the urea-dependent rates of the I<-->N and U<-->N transitions to give the dependence of free energies of the main transition state and of all three (N, I, and U) stable states on urea concentration.

Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer.

The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.