Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins.

The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.     

Probing protein-protein interactions in real time

We have used a prototype small cantilever atomic force microscope to observe, in real time, the interactions between individual protein molecules. In particular, we have observed individual molecules of the chaperonin protein GroES binding to and then dissociating from individual GroEL proteins, which were immobilized on a mica support. This work suggests that the small cantilever atomic force microscope is a useful tool for studying protein dynamics at the single molecule level.     

Affinity chromatography of GroEL chaperonin based on denatured proteins: Role of electrostatic interactions in regulation of GroEL affinity for protein substrates

The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.     

The unfolding action of GroEL on a protein substrate

A molecular dynamics simulation of the active unfolding of denatured rhodanese by the chaperone GroEL is presented. The compact denatured protein is bound initially to the cis cavity and forms stable contacts with several of the subunits. As the cis ring apical domains of GroEL undergo the transition from the closed to the more open (ATP-bound) state, they exert a force on rhodanese that leads to the increased unfolding of certain loops. The contacts between GroEL and rhodanese are analyzed and their variation during the GroEL transition is shown. The major contacts, which give rise to the stretching force, are found to be similar to those observed in crystal structures of peptides bound to the apical domains. The results of the simulation show that multidomain interactions play an essential role, in accord with experiments. Implications of the results for mutation experiments and for the action of GroEL are discussed.     

Chaperonins GroEL and GroES: views from atomic force microscopy.

The Escherichia coli chaperonins, GroEL and GroES, as well as their complexes in the presence of a nonhydrolyzable nucleotide AMP-PNP, have been imaged with the atomic force microscope (AFM). We demonstrate that both GroEL and GroES that have been adsorbed to a mica surface can be resolved directly by the AFM in aqueous solution at room temperature. However, with glutaraldehyde fixation of already adsorbed molecules, the resolution of both GroEL and GroES was further improved, as all seven subunits were well resolved without any image processing. We also found that chemical fixation was necessary for the contact mode AFM to image GroEL/ES complexes, and in the AFM images. GroEL with GroES bound can be clearly distinguished from those without. The GroEL/ES complex was about 5 nm higher than GroEL alone, indicating a 2 nm upward movement of the apical domains of GroEL. Using a slightly larger probe force, unfixed GroEL could be dissected: the upper heptamer was removed to expose the contact surface of the two heptamers. These results clearly demonstrate the usefulness of cross-linking agents for the determination of molecular structures with the AFM. They also pave the way for using the AFM to study the structural basis for the function of GroE system and other molecular chaperones.     

Binding of polylysine to GroEL. Inhibition of the refolding of mMDH.

Luminescence techniques have been used to investigate the interaction of GroEL with polylysine tagged with a fluorescent probe. The fluorescence emitted by anthraniloyl-polylysine, upon excitation at 320 nm, is enhanced by the addition of stoichiometric amounts of GroEL. The equilibrium dissociation constant of the complex (Kd=50 nM) was determined by fluorometric titrations. The rate and extent of recovery of the catalytic activity of denatured mitochondrial malate dehydrogenase, assisted by GroEL, is influenced by either polylysine or anthraniloyl-polylysine. It is suggested that interaction of the positively charged poly-amino acid with the apical domain of GroEL prevents binding of the unfolded protein substrate.     

Single molecular observation of the interaction of GroEL with substrate proteins.

To understand the mechanism of GroEL-assisted protein folding, we observed the interaction of fluorescence-labeled GroEL with fluorescence-labeled substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. GroEL with a A133C mutation in the equatorial domain was labeled with a fluorescent dye, tetramethylrhodamine. As substrate proteins, we used the largely denatured and partly denatured forms of bovine beta-lactoglobulin, both labeled with another fluorescent dye, Cy5. The complexes formed by GroEL with these substrates were characterized by size-exclusion gel chromatography. The recovered complexes were then observed by fluorescence microscopy. For both substrates, agreement of the fluorescent spots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level. Similar observation of macroscopic binding by size-exclusion chromatography and microscopic binding by the fluorescence microscopy was done for the folding intermediate of Cy5-labeled bovine rhodanese. The fluorescence microscopy opens a new avenue for studying the interaction of GroEL with substrate proteins.     

NMR analysis of the binding of a rhodanese peptide to a minichaperone in solution.

A detailed structural analysis of interactions between denatured proteins and GroEL is essential for an understanding of its mechanism. Minichaperones constitute an excellent paradigm for obtaining high-resolution structural information about the binding site and conformation of substrates bound to GroEL, and are particularly suitable for NMR studies. Here, we used transferred nuclear Overhauser effects to study the interaction in solution between minichaperone GroEL(193-335) and a synthetic peptide (Rho), corresponding to the N-terminal alpha-helix (residues 11 to 23) of the mitochondrial rhodanese, a protein whose in vitro refolding is mediated by minichaperones. Using a 60 kDa maltose-binding protein (MBP)-GroEL(193-335) fusion protein to increase the sensitivity of the transferred NOEs, we observed characteristic sequential and mid-range transferred nuclear Overhauser effects. The peptide adopts an alpha-helical conformation upon binding to the minichaperone. Thus the binding site of GroEL is compatible with binding of alpha-helices as well as extended beta-strands. To locate the peptide-binding site on GroEL(193-335), we analysed changes in its chemical shifts on adding an excess of Rho peptide. All residues with significant chemical shift differences are localised in helices H8 and H9. Non-specific interactions were not observed. This indicates that the peptide Rho binds specifically to minichaperone GroEL(193-335). The binding region identified by NMR in solution agrees with crystallographic studies with small peptides and with fluorescence quenching studies with denatured proteins.     

Analysis of interaction between chaperonin GroEL and its substrate using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) provides information about translational diffusion properties of fluorescent molecules in tiny detection volume and allows the analysis of binding processes of biomolecules in homogeneous solution. In this study, FCS was used to measure equilibrium binding constants of disulfide-reduced apo-alpha-lactalbumin (rLA), denatured pepsin, and apo-cytochrome c (apo-cyt c) bound by chaperonin GroEL at different salt concentrations. The results indicate that apo-cyt-c has a much stronger affinity to GroEL than denatured pepsin and rLA have. Titration experiments of GroEL to each substrate with various concentrations of four kinds of salts (K+, Na+, Ca2+, and Mg2+) show that the binding constant of denatured pepsin and rLA to GroEL depends on the salt concentration. The dependence of denatured pepsin binding to GroEL on salt concentration is much stronger than that of rLA. However, the interaction of positively charged apo-cyt c with GroEL is not affected by the salt concentration. Furthermore, the divalent cation promotes the binding of GroEL to denatured pepsin and rLA more strongly than does the monovalent cation.     

Force spectroscopy of collagen fibers to investigate their mechanical properties and structural organization

Tendons are composed of collagen and other molecules in a highly organized hierarchical assembly, leading to extraordinary mechanical properties. To probe the cross-links on the lower level of organization, we used a cantilever to pull substructures out of the assembly. Advanced force probe technology, using small cantilevers (length <20 microm), improved the force resolution into the sub-10 pN range. In the force versus extension curves, we found an exponential increase in force and two different periodic rupture events, one with strong bonds (jumps in force of several hundred pN) with a periodicity of 78 nm and one with weak bonds (jumps in force of <7 pN) with a periodicity of 22 nm. We demonstrate a good correlation between the measured mechanical behavior of collagen fibers and their appearance in the micrographs taken with the atomic force microscope.     

Immunodetection of the recombinant GroEL by the Nanobody NbBruc02

Brucella has a great impact on health and economy in Syria, thus much effort is being placed on the development of diagnostics and vaccines. In this context, a wide Nanobody "immune" library was previously established, from which several Brucella-specific binders were isolated. One of these camel genetically engineered heavy-chain antibody fragments was referred to as NbBruc02. The precise antigen of NbBruc02 was presumed to be, according to proteomic approaches, the Brucella heat shock protein of 60?kDa (HSP-60). HSP-60, or alternatively named GroEL, is an interesting Brucella immunodominant antigen with important roles in the parasite life cycle, mainly adhesion and penetration during the infection of macrophages. In the present work, the capacity of NbBruc02 to filtrate the native GroEL from Brucella total extract was tested by immunochromatography approach. The interaction between NbBruc02 and its antigen was further confirmed using recombinant GroEL from Brucella. Interestingly, NbBruc02 was able to immunodetect the native as well as the denatured forms of the rGroEL in ELISA and immunoblotting, respectively. In agreement with previously reported data, NbBruc02 was able only to detect the denatured Yersinia rGroEL. Using surface plasmon resonance (SPR) biosensor, NbBruc02 showed a strong interaction, with nanomolar affinity (K (D)?=?~10(-8)?M), with the native rGroEL of Brucella and not of Yersinia. Because the casual conformational changes in the GroEL 3D structure make the base of its function, NbBruc02 by its ability to recognize a "conformational epitope," could open wide perspectives to study the role of GroEL in Brucella physiology.     

GroEL and protein disulfide isomerase each binds with folding intermediates of D-glyceraldehyde-3-phosphate dehydrogenase released from complexes formed with the other

Simultaneous presence of two chaperones, GroEL and protein disulfide isomerase (PDI), assists the reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in an additive way. Delayed addition of chaperones to the refolding solution after dilution of denatured GAPDH indicates an interaction with intermediates formed mainly in the first 5 min for PDI and formed within a longer time period for GroEL-ATP. The above indicate that the two chaperones interact with different folding intermediates of GAPDH. After delayed addition of one chaperone to the refolding mixture containing the other at 4°C, GroEL binds with all GAPDH intermediates dissociated from PDI, and PDI interacts with the intermediates released from GroEL during the first 10–20 min. It is suggested that the GAPDH folding intermediates released from the chaperone-bound complex are still partially folded so as to be rebound by the other chaperone. The above results clearly support the network model of GroEL and PDI.     

Differentiating inclusion complexes from host molecules by tapping-mode atomic force microscopy.

Tapping-mode atomic force microscopy imaging under different cantilever vibration amplitudes has been used to differentiate the host beta-cyclodextrin nanotubes from retinal/beta-cyclodextrin inclusion complex nanotubes. It was observed that both compounds were deformed differently by the applied probe force because of their different local rigidity. This change in the elasticity properties can be explained as a consequence of the inclusion process. This method shows that tapping-mode atomic force microscopy is an useful tool to map soft sample elasticity properties and to distinguish inclusion complexes from their host molecules on the basis of their different mechanical response.     

Non-functioning chaperonin GroEL stimulates protein aggregation

To clarify the role of chaperones in the development of amyloid diseases, the interaction of the chaperonin GroEL with misfolded proteins and recombinant prions has been studied. The efficiency of the chaperonin-assisted folding of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to be decreased in the presence of prions. Prions are capable of binding to GroEL immobilized on Sepharose, but this does not prevent the interaction between GroEL and other denatured proteins. The size of individual proteins (GroEL, GAPDH, and the recombinant prion) and aggregates formed after their mixing have been determined by the dynamic light scattering analysis. It was shown that at 25°C, the non-functioning chaperonin (equimolar mixture of GroEL and GroES in the absence of Mg-ATP) bound prion yielding large aggregates (greater than 400 nm). The addition of Mg-ATP decreased significantly the size of the aggregates to 70–80 nm. After blocking of one of the chaperonin active sites by oxidized denatured GAPDH, the aggregate size increased to 1200 nm, and the addition of Mg-ATP did not prevent the aggregation. These data indicate the significant role of chaperonins in the formation of amyloid structures and demonstrate the acceleration of aggregation in the presence of functionally inactive chaperonins. The suggested model can be used for the analysis of the efficiency of antiaggregants in the system containing chaperonins.     

Identification of substrate binding site of GroEL minichaperone in solution.

It is difficult to obtain high-resolution structural information on the substrate-binding site of intact GroEL. But minichaperones, domains containing the peptide-binding site of GroEL, do constitute tractable systems for detailed studies. A peptide-binding site was located in crystals of a minichaperone and proposed to constitute a model for substrate-binding. We have now located the substrate binding site of the minichaperone GroEL(193-335) in solution by labelling it at various positions with a fluorescent probe and detecting which positions are perturbed on binding a denatured substrate. The fluorescence of a probe attached to a cysteine residue engineered at position 228 (N terminus of helix H8), 241 (helix H8), 261 (helix H9), or 267 (helix H9) was affected significantly by binding of substrate. But there was little change for a label at positions 193, 212, 217 or 293. The dissociation constants between substrates and minichaperone were evaluated from fluorescence anisotropy assays. The effects of salt and temperature were the same as those with intact GroEL. These results indicate that the region around helices H8 and H9 is the substrate-binding site for the apical domain fragment. Intriguingly, the same site is involved in the binding of GroES. Thus, an important function of GroES in the regulation of the activity of GroEL for substrates is to displace the bound substrate by competing for its binding site.     

Separation of copurifying GroEL from glutathione-S-transferase fusion proteins

The purification of overexpressed fusion proteins using bacterial expression systems is a useful tool for the study of many proteins. One problem that can occur is the formation of stable interactions between the expressed fusion protein and certain endogenous bacterial proteins, such as the molecular chaperone GroEL. Such interactions may result in the copurification of contaminating bacterial proteins. Here we describe an efficient and inexpensive method for the removal of contaminating GroEL from a bacterially expressed GST fusion protein. In this method, denatured bacterial proteins are added to the bacterial lysates prior to the addition of glutathione Sepharose resin. The denatured proteins compete for GroEL binding, thereby releasing the GroEL contaminants from the expressed fusion protein.     

Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL

In order to fold non-native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP-dependent reaction cycle. We constructed the real-time three-dimensional-observation system at high resolution using a newly developed fast-scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s(-1)). We also caught ATP/ADP-induced open-closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP-bound GroEL can change its conformation 'from closed to open' without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP ( approximately 1.0 s) was apparently lower than those of ATP and ATP-analogs (2-3 s), meaning that ADP-bound open-form is structurally less stable than ATP-bound open-form. These results indicate that GroEL has at least two distinct open-conformations in the presence of nucleotide; ATP-bound prehydrolysis open-form and ADP-bound open-form, and the ATP hydrolysis in open-form destabilizes its open-conformation and induces the 'from open to closed' conformational change of GroEL.     

Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.

The mechanism of GroEL (chaperonin)-mediated protein folding is only partially understood. We have analysed structural and functional properties of the interaction between GroEL and the co-chaperonin GroES. The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo-chaperonin is 1:1. GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C-terminal residues. Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full-length GroEL. Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder. This apparently prohibits the association of a second GroES oligomer. Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain-excluding masses within the GroEL double-ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa). We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL.     

Imaging material properties of biological samples with a force feedback microscope

Mechanical properties of biological samples have been imaged with a force feedback microscope. Force, force gradient, and dissipation are measured simultaneously and quantitatively, merely knowing the atomic force microscopy cantilever spring constant. Our first results demonstrate that this robust method provides quantitative high resolution force measurements of the interaction. The small oscillation imposed on the cantilever and the small value of its stiffness result in vibrational energies much smaller than the thermal energy, reducing interaction with the sample to a minimum. We show that the observed mechanical properties of the sample depend on the force applied by the tip and consequently on the sample indentation. Copyright © 2013 John Wiley & Sons, Ltd.     

Dense chromatin plates in metaphase chromosomes

In a previous work we observed multilayered plate-like structures surrounding partially denatured HeLa chromosomes at metaphase ionic conditions. This unexpected finding has led us to carry out an extensive investigation of these structures. Our results show that plates can also be found in metaphase chromosomes from chicken lymphocytes. We have used atomic force microscopy (AFM) to image and investigate the mechanical properties of plates in aqueous solution. Plates are thin (~6.5 nm each layer) but compact and resistant to penetration by the AFM tip: their Young’s modulus is ~0.2 GPa and the stress required for surface penetration is ~0.03 GPa in the presence of Mg²⁺ (5–20 mM). Low-ionic strength conditions produce emanation of chromatin fibers from the edges of uncrosslinked plates. These observations and AFM results obtained applying high forces indicate that the chromatin filament is tightly tethered inside the plates. Images of metal-shadowed plates and cryo-electron microscopy images of frozen-hydrated plates suggest that nucleosomes are tilted with respect to the plate surface to allow an interdigitation between the successive layers and a thickness reduction compatible with the observed plate height. The similarities between denatured plates from chicken chromosomes and aggregates of purified chromatin from chicken erythrocytes suggest that chromatin has intrinsic structural properties leading to plate formation. Scanning electron micrographs and images obtained with the 200-kV transmission microscope show that plates are the dominant component of compact chromatids. We propose that metaphase chromosomes are formed by many stacked plates perpendicular to the chromatid axis.