Domain-specific chaperone-induced expansion is required for beta-actin folding: a comparison of beta-actin conformations upon interactions with GroEL and tail-less complex polypeptide 1 ring complex (TRiC)

Actin, an abundant cytosolic protein in eukaryotic cells, is dependent on the interaction with the chaperonin tail-less complex polypeptide 1 ring complex (TRiC) to fold to the native state. The prokaryotic chaperonin GroEL also binds non-native beta-actin, but is unable to guide beta-actin toward the native state. In this study we identify conformational rearrangements in beta-actin, by observing similarities and differences in the action of the two chaperonins. A cooperative collapse of beta-actin from the denatured state to an aggregation-prone intermediate is observed, and insoluble aggregates are formed in the absence of chaperonin. In the presence of GroEL, however, >90% of the aggregation-prone actin intermediate is kept in solution, which shows that the binding of non-native actin to GroEL is effective. The action of GroEL on bound flourescein-labeled beta-actin was characterized, and the structural rearrangement was compared to the case of the beta-actin-TRiC complex, employing the homo fluorescence resonance energy transfer methodology previously used [Villebeck, L., Persson, M., Luan, S.-L., Hammarstr?m, P., Lindgren, M., and Jonsson, B.-H. (2007) Biochemistry 46 (17), 5083-93]. The results suggest that the actin structure is rearranged by a "binding-induced expansion" mechanism in both TRiC and GroEL, but that binding to TRiC, in addition, causes a large and specific separation of two subdomains in the beta-actin molecule, leading to a distinct expansion of its ATP-binding cleft. Moreover, the binding of ATP and GroES has less effect on the GroEL-bound beta-actin molecule than the ATP binding to TRiC, where it leads to a major compaction of the beta-actin molecule. It can be concluded that the specific and directed rearrangement of the beta-actin structure, seen in the natural beta-actin-TRiC system, is vital for guiding beta-actin to the native state.     

Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits.

T-complex polypeptide 1 (TCP-1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP-1 to be part of a hetero-oligomeric 970 kDa complex containing several structurally related subunits of 52-65 kDa. These members of a new protein family are assembled into a TCP-1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP-dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co-chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.     

The t complex polypeptide 1 (TCP-1) is associated with the cytoplasmic aspect of Golgi membranes

The t complex polypeptide 1 (TCP-1) is a protein of unknown function expressed in large amounts during spermatogenesis. Rat monoclonal antibodies recognizing TCP-1 have been prepared and used to immunoprecipitate and Western blot a 57 kd protein from germ cell and tissue culture cell extracts. In tissue culture cells, indirect immunofluorescent localization of antigen indicated a perinuclear distribution similar to that of the Golgi apparatus. Analysis of the TCP-1 distribution in tissue culture cells showed that the polypeptide was associated with the cytoplasmic aspect of membranes of the trans-Golgi network (TGN). The distribution in spermatids suggested that TCP-1 was localized to structures often associated with the developing acrosome. The TCP-1 antigenic epitopes are highly conserved, allowing the protein to be identified in cells across a wide variety of vertebrate species and tissues. These experiments suggest that TCP-1 may be essential for transport of proteins through the exocytic pathway in all cells and required in large amounts for acrosome formation in developing spermatids.     

Brain t-complex polypeptide 1 (TCP- 1) related to its natural substrate beta1 tubulin is decreased in Alzheimer's disease.

The t-complex polypeptide 1 is a selective molecular chaperone in tubulin biogenesis, by that nascent tubulin subunits are bound to t-complex polypeptide 1 and released in assembly competent forms. In neurodegenerative diseases with Alzheimer pathology cytoskeletal proteins are deficient and aggregated. Therefore we examined t-complex polypeptide 1 as represented by the zeta subunit and its specific substrate beta 1 tubulin represented by a truncated product in six brain regions of nine patients with Alzheimer's disease, nine patients with Down syndrome and nine controls. We used 2 dimensional electrophoresis with in-gel-digestion and matrix-assisted laser desorption/ ionization- mass spectrometry for the separation and identification of human brain t-complex polypeptide 1 and beta 1 tubulin. When t-complex polypeptide I was related to its natural and specific substrate beta 1 tubulin, the ratio was significantly decreased in the temporal, frontal, parietal cortex and in thalamus of patients with Alzheimer's disease. In Down syndrome the t-complex polypeptide 1/beta 1 tubulin ratio was significantly increased in frontal and parietal cortex suggesting a different mechanism for aggregation of microfilament proteins e.g. beta 1 tubulin. Relatively decreased molecular chaperoning of beta 1 tubulin by t-complex polypeptide 1 may lead to misfolded tubulin aggregating and accumulating in plaques and tangles, a hallmark of Alzheimer's disease. Our contribution provides first clues for a mechanism of microtubular accumulation in Alzheimer's disease and challenges further studies on different chaperones and chaperonins in the brain of patients with neurodegenerative diseases.     

The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction

Sperm-oocyte interactions are among the most remarkable processes in cell biology. These cellular recognition events are initiated by an exquisitely specific adhesion of free-swimming spermatozoa to the zona pellucida, an acellular matrix that surrounds the ovulated oocyte. Decades of research focusing on this interaction have led to the establishment of a widely held paradigm that the zona pellucida receptor is a single molecular entity that is constitutively expressed on the sperm cell surface. In contrast, we have employed the techniques of blue native-polyacrylamide gel electrophoresis, far Western blotting, and proximity ligation to secure the first direct evidence in support of a novel hypothesis that zona binding is mediated by multimeric sperm receptor complex(es). Furthermore, we show that one such multimeric association, comprising the chaperonin-containing TCP1 complex (CCT/TRiC) and a zona-binding protein, zona pellucida-binding protein 2, is present on the surface of capacitated spermatozoa and could account for the zona binding activity of these cells. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize.     

Conformational distributions and proximity relationships in the rigor complex of actin and myosin subfragment-1

Cyclic conformational changes in the myosin head are considered essential for muscle contraction. We hereby show that the extension of the fluorescence resonance energy transfer method described originally by Taylor et al. (Taylor, D. L., Reidler, J., Spudich, J. A., and Stryer, L. (1981) J. Cell Biol. 89, 362-367) allows determination of the position of a labeled point outside the actin filament in supramolecular complexes and also characterization of the conformational heterogeneity of an actin-binding protein while considering donor-acceptor distance distributions. Using this method we analyzed proximity relationships between two labeled points of S1 and the actin filament in the acto-S1 rigor complex. The donor (N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate) was attached to either the catalytic domain (Cys-707) or the essential light chain (Cys-177) of S1, whereas the acceptor (5-(iodoacetamido)fluorescein) was attached to the actin filament (Cys-374). In contrast to the narrow positional distribution (assumed as being Gaussian) of Cys-707 (5 +/- 3 A), the positional distribution of Cys-177 was found to be broad (102 +/- 4 A). Such a broad positional distribution of the label on the essential light chain of S1 may be important in accommodating the helically arranged acto-myosin binding relative to the filament axis.     

FISH-microdissection (FISH-MD) analysis of complex chromosome rearrangements

We combined the techniques of fluorescence in situ hybridization (FISH) and chromosomal microdissection in one experiment (FISH-MD). This novel method permits rapid identification of the composition, origin, and breakpoints of rearranged chromosomes. Rearranged chromosomes are first identified by multicolor-FISH, then the fluorophore-labeled derivative chromosomes are directly isolated by microdissection and reverse painted to identify the breakpoints.     

1H-NMR studies on poly(oxyethylene)-bound oligopeptides

Conformational studies on poly(oxyethylene)-bound homo-, oligo-, guest-host, and sequential peptides synthesized according to the liquid-phase method were carried out by means of ¹H-nmr spectroscopy. The solubilizing effect of the C-terminal polymeric support allowed a thorough investigation of the secondary structure in solution.     

Conformational instability of the cholera toxin A1 polypeptide

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by vesicular transport. In the ER, the catalytic CTA1 subunit dissociates from the holotoxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). It is hypothesized that CTA1 triggers its ERAD-mediated translocation into the cytosol by masquerading as a misfolded protein, but the process by which CTA1 activates the ERAD system remains unknown. Here, we directly assess the thermal stability of the isolated CTA1 polypeptide by biophysical and biochemical methods and correlate its temperature-dependent conformational state with susceptibility to degradation by the 20S proteasome. Measurements with circular dichroism and fluorescence spectroscopy demonstrated that CTA1 is a thermally unstable protein with a disordered tertiary structure and a disturbed secondary structure at 37 °C. A protease sensitivity assay likewise detected the temperature-induced loss of native CTA1 structure. This protease-sensitive conformation was not apparent when CTA1 remained covalently associated with the CTA2 subunit. Thermal instability in the dissociated CTA1 polypeptide could thus allow it to appear as a misfolded protein for ERAD-mediated export to the cytosol. In vitro, the disturbed conformation of CTA1 at 37 °C rendered it susceptible to ubiquitin-independent degradation by the core 20S proteasome. In vivo, CTA1 was also susceptible to degradation by a ubiquitin-independent proteasomal mechanism. ADP-ribosylation factor 6, a cytosolic eukaryotic protein that enhances the enzymatic activity of CTA1, stabilized the heat-labile conformation of CTA1 and protected it from in vitro degradation by the 20S proteasome. Thermal instability in the reduced CTA1 polypeptide has not been reported before, yet both the translocation and degradation of CTA1 may depend upon this physical property.     

Chaperonin-containing TCP-1 complex directly binds to the cytoplasmic domain of the LOX-1 receptor

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a scavenger receptor that binds oxidized low-density lipoprotein (OxLDL) and has a role in atherosclerosis development. The N-terminus intracellular region (cytoplasmic domain) of LOX-1 mediates receptor internalization and trafficking, potentially through intracellular protein interactions. Using affinity isolation, we identified 6 of the 8 components of the chaperonin-containing TCP-1 (CCT) complex bound to LOX-1 cytoplasmic domain, which we verified by coimmunoprecipitation and immunostaining in human umbilical vein endothelial cells. We found that the interaction between CCT and LOX-1 is direct and ATP-dependent and that OxLDL suppressed this interaction. Understanding the association between LOX-1 and the CCT complex may facilitate the design of novel therapies for cardiovascular disease.     

Conformational aspect of polypeptide structure. XLIV. Conformational transitions of poly(N-methyl-alanines) induced by trifluoroacetic acid

The synthesis of poly(N-methyl-L -alanine) and poly (N-methyl-DL -alanine) are described. The polymers were examined by 220 MHz high-resolution nuclear magnetic resonance (nmr) and circular dichroism (CD). The results demonstrate that poly(N-methyl-L -alanine) exists as an ordered helical structure with all the amide bonds in the trans configuration in appropriate solvents. As trifluoroacetic acid (TFA) is added to the solutions of the polymer in helix-supporting solvents, resonances corresponding to both trans and cis amide conformations of N-methyl, C-methyl, and α-CH are observed. The presence of both the trans and the cis peptide bonds in a polymer chain disrupts the ordered structures. Our conclusions from CD data are in agreement with the nmr results. Ultracentrifugation shows that degradation of the polymer chain does not occur during the TFA treatment.     

Conformational transitions of islet amyloid polypeptide (IAPP) in amyloid formation in vitro

Amyloid aggregates have been recognized to be a pathological hallmark of several fatal diseases, including Alzheimer's disease, the prion-related diseases, and type II diabetes. Pancreatic amyloidosis is characterized by the deposition of amyloid consisting of islet amyloid polypeptide (IAPP). We followed the steps preceding IAPP insolubilization and amyloid formation in vitro using a variety of biochemical methods, including a filtration assay, far and near-UV circular dichroism (CD) spectropolarimetry, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, and atomic force (AFM) and electron (EM) microscopy. IAPP insolubilization and amyloid formation followed kinetics that were consistent with the nucleation-dependent polymerization mechanism. Nucleation of IAPP amyloid formation with traces of preformed fibrils induced a rapid conformational transition into beta-sheets that subsequently aggregated into insoluble amyloid fibrils. Transition proceeded via a molten globule-like conformeric state with large contents of secondary structure, fluctuating tertiary and quaternary aromatic interactions, and strongly solvent-exposed hydrophobic patches. In the temperature denaturation pathway at 5 microM peptide, we found that this state was mostly populated at about 45 degrees C, and either aggregated rapidly into amyloid by prolonged exposure to this temperature, or melted into denaturated but still structured IAPP, when heated further to 65 degrees C. The state at 45 degrees C was also found to be populated at 4.25 M GdnHCl at 25 degrees C during GdnHCl-induced equilibrium denaturation, and was stable in solution for several hours before aggregating into amyloid fibrils. Our studies suggested that this amyloidogenic state was a self-associated form of an aggregation-prone, partially folded state of IAPP. We propose that this partially folded population and its self-associated forms are in a concentration-dependent equilibrium with a non-amyloidogenic IAPP conformer and may act as early, soluble precursors of beta-sheet and amyloid formation. Our findings on the molecular mechanism of IAPP amyloid formation in vitro should assist in gaining insight into the pathogenesis and inhibition of pancreatic amyloidosis and other amyloid-related diseases.     

Conformational study of asialo-GM1 (GA1) ganglioside

In order to investigate the significance of preferred conformations of the saccharide for the steric orientation and recognition of glycosphingolipids at the membrane surface, the conformational free energy calculations were carried out on the asialo-GM1 [GA1; β-D -Gal(1 → 3) β-D -GalNAc(1 → 4) β-D -Gal(1 → 4) β-D -Glc-O-ceramide] using a new program CONCARB (CONformational study program for CARBohydrate) in the unhydrated and hydrated states. The overall backbone conformation of GA1 appears nearly to be extended with a little bent at the glycosidic II–III linkage, in which two pyranose rings of Gal(IV)-GalNAc-(III) moiety orient approximately perpendicular to those of Gal(II)-Glc(I) moiety. This is consistent with the structures deduced from high-sensitivity differential scanning calorimetry experiments and the nmr study on GA1. The calculated glycosidic torsion angles of the lowest free energy conformation of GA1 in the hydrated state are in accord with the structures of relevant oligosaccharides deduced from nmr experiments and hard sphere exoanomeric calculations. A comparison of the values of glycosidic torsion angles ϕ and π of GA1 and its constituent oligosaccharides indicates that the overall backbone conformation of each oligosaccharide is retained when the oligosaccharide chain becomes longer. This implies that the short-range interactions between the nearest-neighbored saccharides are of significant importance in stabilizing the overall backbone conformation of GA1 in both the unhydrated and hydrated states. The different orientation and hydrogen bonds of hydroxymethyl and hydroxyl groups from one oligosaccharide to another suggest that the medium- and long-range interactions are also of consequence. Hydration seems to affect significantly the conformation of these groups, but not to perturb remarkably the overall backbone conformation of GA1. © 1997 John Wiley & Sons, Inc. Biopoly 42: 19–35, 1997     

Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, studied with formycin nucleotides.

1. Fluorescence measurements have shown that formycin triphosphate (FTP) or formycin diphosphate (FDP) bound to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in Na+-containing media can be displaced by the following ions (listed in order of effectiveness): Tl+, K+, Rb+, NH4+, Cs+. 2. The differences between the nucleotide affinities displayed by the enzyme in predominantly Na+ and predominantly K+ media in the absence of phosphorylation, are thought to reflect changes in enzyme conformation. These changes can therefore be monitored by observing the changes in fluorescence that accompany net binding or net release of formycin nucleotides. 3. The transition from a K+-bound form (E2-(K)) to an Na+-bound form (E1-Na) is remarkably slow at low nucleotide concentrations, but is accelerated if the nucleotide concentration is increased. This suggests that the binding of nucleotide to a low-affinity site on E2-(K) accelerates its conversion to E1-Na; it supports the hypothesis that during the normal working of the pump, ATP, acting at a low affinity site, accelerates the conversion of dephosphoenzyme, newly formed by K+-catalysed hydrolysis of E2P, to a form in which it can be phosphorylated in the presence of Na+. 4. The rate of the reverse transformation, E1-Na to E2-(K), varies roughly linearly with the K+ concentration up to the highest concentration at which the rate can be measured (15 mM). Since much lower concentrations of K+ are sufficient to displace the equilibrium to the K-form, we suggest that the sequence of events is: (i) combination of K+ with low affinity (probably internal) binding sites, followed by (ii) spontaneous conversion of the enzyme to a form, E2-(K), containing occluded K+. 5. Mg2+ or oligomycin slows the rate of conversion of E1-Na to E2-(K) but does not significantly affect the rate of conversion of E2-(K) to E1-Na. 6. In the light of these and previous findings, we propose a model for the sodium pump in which conformational changes alternate with trans-phosphorylations, and the inward and outward fluxes of both Na+ and K+ each involve the transfer of a phosphoryl group as well as a change in conformation between E1 and E2 forms of the enzyme or phosphoenzyme.     

Cloning of a cDNA encoding the Tcp-1 (t complex polypeptide 1) homologue of Arabidopsis thaliana.

We isolated a plant homologue of Tcp-1 (t complex polypeptide 1) cDNA from Arabidopsis thaliana. It encodes a 545-amino acid (aa) protein, which has extensive aa and nucleotide sequence homology to the corresponding proteins of mouse, yeast, fruit fly and archaebacteria.     

Conformational changes of S-1 related to its dissociation from actin.

The peptide pattern obtained after proteolysis of S-1 with trypsin was different in the absence or presence of anions. The affinity of tryptic and undigested S-1 for anions (CN-, SCN- or HCO3-) was different, as reflected by the altered values of Ki or Ka obtained from ATPase activity measurements. Anions CN-, SCN-, HCO3-, or PPi induced dissociation of actomyosin when added to acto-S-1 or acto-heavy-meromyosin. Among nucleoside di- and triphosphates, only triphosphates were effective with regard to the dissociation. The results suggest the existence of a regulatory site of cationic nature on S-1, which might be involved in the dissociation of actin from myosin.     

Conformational properties of the main intrinsic polypeptide (MIP26) isolated from lens plasma membranes

The conformational properties of the main intrinsic polypeptide (MIP26) isolated from lens plasma membranes were studied by using near- and far-ultraviolet circular dichroism. The far-ultraviolet spectrum of MIP26 solubilized with octyl beta-D-glucopyranoside indicates an alpha-helical content of approximately 50% and a beta-structure content of approximately 20%. A detergent-free membrane suspension of MIP26 produced a typically distorted far-ultraviolet spectrum which was caused by differential light scattering and absorption flattening. However, decreasing the size of the membrane fragments by sonication produced a far-ultraviolet spectrum free of distortion, and with a rotatory strength profile similar to that obtained for MIP26 solubilized with octyl beta-D-glucopyranoside. This implies similar secondary structure properties for the protein in both the suspension and the sugar detergent. The cleavage of MIP26 with Staphylococcus aureus protease, which results in removal of a 5-kilodalton peptide and which mimics the age-dependent posttranslational changes that take place in the lens, did not significantly affect the conformation of the core protein as judged by the near-ultraviolet circular dichroism spectra.     

Synthesis of ring halogenated prostaglandins (1)

The synthesis of nine mono- and difluoro prostaglandins , , , , , , and , and two monochloro prostaglandins and , from appropriately protected derivatives of natural PGF₂ is described.     

Conformational changes of actin induced by strong or weak myosin subfragment-1 binding

Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the back surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction.