Tetramethylrhodamine Dimer Formation as a Spectroscopic Probe of the Conformation of Escherichia coli Ribosomal Protein L7/L12 Dimers

Hamman, Brian D.; Oleinikov, Andrew V.; Jokhadze, George G.; Bochkariov, Dmitry; Traut, Robert R.; Jameson, David M.

doi:10.1074/jbc.271.13.7568

Cited by 55 publications

(78 citation statements)

References 27 publications

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“…This shift between the rhodamine dimer and monomer populations is indicated by the progressive change in the OD 518 ͞ OD 555 ratio (Fig. 2b Inset), which corresponds to the dimer͞ monomer ratio (36). For TMRIA, an OD 518 ͞OD 555 range of 0.4-1.3 has been reported (36), with 1.3 and 0.4 corresponding to the purely dimeric and monomeric forms, respectively.…”

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confidence: 84%

Global configuration of single titin molecules observed through chain-associated rhodamine dimers

Grama

Somogyi

Kellermayer

2001

Proc. Natl. Acad. Sci. U.S.A.

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The global configuration of individual, surface-adsorbed molecules of the giant muscle protein titin, labeled with rhodamine conjugates, was followed with confocal microscopy. Fluorescence-emission intensity was reduced because of self-quenching caused by the close spacing between rhodamine dye molecules that formed dimers. In the presence of chemical denaturants, fluorescence intensity increased, reversibly, up to 5-fold in a fast reaction; the kinetics were followed at the single-molecule level. We show that dimers formed in a concentrated rhodamine solution dissociate when exposed to chemical denaturants. Furthermore, titin denaturation, followed by means of tryptophan fluorescence, is dominated by a slow reaction. Therefore, the rapid fluorescence change of the single molecules reflects the direct action of the denaturants on rhodamine dimers rather than the unfolding͞refolding of the protein. Upon acidic denaturation, which we have shown not to dissociate rhodamine dimers, fluorescence intensity change was minimal, suggesting that dimers persist because the unfolded molecule has contracted into a small volume. The highly contractile nature of the acid-unfolded protein molecule derives from a significant increase in chain flexibility. We discuss the potential implications this finding could have for the passive mechanical behavior of striated muscle.T itins are 3.0-3.7 MDa filamentous proteins extending between the Z-and M-lines of the sarcomere (1-4). Titin is a tandem array of Ig type C2 and fibronectin (FN) type III domains (Ϸ300 per molecule) interspersed with unique sequences, most notably the Pro-, Glu-, Val-, and Lys-rich PEVK segment, and the N2A and N2B segments (5). The I-band segment of titin acts as a molecular spring, whose elastic properties define the passive or restoring mechanical properties of striated muscle (6-9). By contrast, titin's A-band segment is thought to function as a scaffold that defines structural regularity within the A band (10). Recent single-molecule experiments often have used titin as a model system because of a large size that makes it relatively easily accessible to such investigations. Single-molecule-mechanics experiments using either optical tweezers (11-13) or atomic force microscopy (AFM; ref. 14) described titin as an entropic chain in which domain unfolding occurs at high forces and refolding occurs at low forces. These experiments formed the basis of many theoretical and modeling works that addressed the mechanisms of mechanically driven protein folding (15)(16)(17)(18)(19)(20). Individual titin molecules can be made visible under aqueous conditions after labeling with fluorophores. Fluorescently labeled titin molecules have recently been studied under various conditions: equilibration to a surface (21-23), stretching with meniscus force (22, 23) and direct manipulation (24), and chemical denaturation (25,26). Chemical denaturation has been shown to cause an abrupt and significant rise in the fluorescence intensity of Oregon Green 488 (Molecular Probes) maleimi...

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confidence: 84%

Global configuration of single titin molecules observed through chain-associated rhodamine dimers

Grama

Somogyi

Kellermayer

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…It is often covalently attached using a thiol-or aminereactive linker group. As in previous studies (15)(16)(17), we took advantage of the dimer formation or stacking propensity of TMR. Dimer formation is accompanied by a change in the absorbance and fluorescence properties of the dye, a deviation that can be explained by Kasha's exciton coupling model (18).…”

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confidence: 99%

Measuring how much work the chaperone GroEL can do

Corsepius

Lorimer

2013

Proc. Natl. Acad. Sci. U.S.A.

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Noncovalently "stacked" tetramethylrhodamine (TMR) dimers have been used to both report and perturb the allosteric equilibrium in GroEL. A GroEL mutant (K242C) has been labeled with TMR, close to the peptide-binding site in the apical domain, such that TMR molecules on adjacent subunits are able to form dimers in the T allosteric state. Addition of ATP induces the transition to the R state and the separation of the peptide-binding sites, with concomitant unstacking of the TMR dimers. A statistical analysis of the spectra allowed us to compute the number and orientation of TMR dimers per ring as a function of the average number of TMR molecules per ring. The TMR dimers thus serve as quantitative reporter of the allosteric state of the system. The TMR dimers also serve as a surrogate for substrate protein, substituting in a more homogeneous, quantifiable manner for the heterogeneous intersubunit, intraring, noncovalent crosslinks provided by the substrate protein. The characteristic stimulation of the ATPase activity by substrate protein is also mimicked by the TMR dimers. Using an expanded version of the nested cooperativity model, we determine values for the free energy of the TT to TR and TR to RR allosteric equilibria to be 27 ± 11 and 46 ± 2 kJ/mol, respectively. The free energy of unstacking of the TMR dimers was estimated at 2.6 ± 1.0 kJ/mol dimer. These results demonstrate that GroEL can perform work during the T to R transition, supporting the iterative annealing model of chaperonin function.allostery | substrate protein binding problem

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“…Both L7/L12 and P1/P2 have in common their size (around 110 -120 residues), their acidity, and the fact that Nand C-terminal domains are joined by an alanine-rich flexible region (15). The C-terminal protruding region (16,17) is identical in P0, P1, and P2 and contains phosphorylation sites not found in prokaryotes. In the rat, P2 phosphorylation has been shown to stimulate the proteosynthetic activity of the ribosome (18) and the GTPase activity of eEF-2 (19).…”

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Pivotal Role of the P1 N-terminal Domain in the Assembly of the Mammalian Ribosomal Stalk and in the Proteosynthetic Activity

Gonzalo

Lavergne

Reboud

2001

Journal of Biological Chemistry

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In the 60 S ribosomal subunit, the lateral stalk made of the P-proteins plays a major role in translation. It contains P0, an insoluble protein anchoring P1 and P2 to the ribosome. Here, rat recombinant P0 was overproduced in inclusion bodies and solubilized in complex with the other P-proteins. This method of solubilization appeared suitable to show protein complexes and revealed that P1, but not P2, interacted with P0. Furthermore, the use of truncated mutants of P1 and P2 indicated that residues 1-63 in P1 connected P0 to residues 1-65 in P2. Additional experiments resulted in the conclusion that P1 and P2 bound one another, either connected with P0 or free, as found in the cytoplasm. Accordingly, a model of association for the P-proteins in the stalk is proposed. Recombinant P0 in complex with phosphorylated P2 and either P1 or its (1-63) domain efficiently restored the proteosynthetic activity of 60 S subunits deprived of native P-proteins. Therefore, refolded P0 was functional and residues 1-63 only in P1 were essential. Furthermore, our results emphasize that the refolding principle used here is worth considering for solubilizing other insoluble proteins.The ribosome is the central constituent of the protein synthesis machinery (1). During translation of messenger RNA into protein, the ribosome is helped by several soluble factors that operate in a sequential manner to improve both efficiency and fidelity of this process (2, 3). How this extraordinary coordination is performed is not yet exactly understood. Still, because most of the translation factors are GTPases, the driving of the factors by the ribosome is likely to be controlled by GTP hydrolysis (4). A small portion of the 28 S rRNA designated as the GTPase center is known to be involved in GTP hydrolysis activation (5). The GTPase center is connected directly to the stalk (6), an elongated and very flexible protuberance interacting with elongation factors (7-10). However, despite decades of research, the organization and functions of the proteins constituting the stalk remain unclear. The number and the nature of the proteins constituting the stalk are different depending on the biological system, although their general organization, made of five proteins, is likely to be similar. In prokaryotes, four identical proteins (L7/L12) are linked to the GTPase center by L10 connected itself with a sixth protein, L11 (11). In mammals, the equivalents of the four L7/L12s are two different proteins, P1 and P2, each being present in two copies. These proteins are bound to P0, the equivalent of L10, which is itself bound to L12, the eukaryotic equivalent of L11, and to the GTPase center (6, 12). In plants, an additional protein, P3, has been described (13). In yeast, there are two variants of both P1 (P1␣ and P1␤) and P2 (P2␣ and P2␤); the precise repartition and function of each variant remains unsettled (14). This structural heterogeneity seems to correspond to functional differences, and data obtained in one system cannot be extrapolated directly (10). Bot...

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Tetramethylrhodamine Dimer Formation as a Spectroscopic Probe of the Conformation of Escherichia coli Ribosomal Protein L7/L12 Dimers

Cited by 55 publications

References 27 publications

Global configuration of single titin molecules observed through chain-associated rhodamine dimers

Global configuration of single titin molecules observed through chain-associated rhodamine dimers

Measuring how much work the chaperone GroEL can do

Pivotal Role of the P1 N-terminal Domain in the Assembly of the Mammalian Ribosomal Stalk and in the Proteosynthetic Activity

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