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Measuring How Much Work the Chaperone Groel Can Do

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Measuring How Much Work the Chaperone Groel Can Do Measuring how much work the chaperone GroEL PNAS PLUS can do Nicholas C. Corsepiusa,b,c and George H. Lorimera,b,c,d,1 aCenter for Biomolecular Structure and Organization, bBiochemistry PhD Program, cDepartment of Chemistry and Biochemistry, and dInstitute for Physical Science and Technology, University of Maryland, College Park, MD 20742 Contributed by George H. Lorimer, April 26, 2013 (sent for review April 1, 2013) SEE COMMENTARY Noncovalently “stacked” tetramethylrhodamine (TMR) dimers have rotation of the apical domains, the expansion of the central been used to both report and perturb the allosteric equilibrium in cavity, and the separation of the peptide binding sites (8). The R GroEL. A GroEL mutant (K242C) has been labeled with TMR, close to state has a high affinity for nucleotide, low affinity for SP, and is the peptide-binding site in the apical domain, such that TMR mole- the acceptor state for GroES. ATP binding is positively coopera- cules on adjacent subunits are able to form dimers in the T allosteric tive between subunits within a ring and negatively cooperative state. Addition of ATP induces the transition to the R state and the between rings within a 14mer. In the absence of GroES, GroEL separation of the peptide-binding sites, with concomitant unstacking can adopt three distinct allosteric states: TT, TR, and RR. of the TMR dimers. A statistical analysis of the spectra allowed us to In the presence of Mg2+ and K+, GroEL hydrolyzes ATP to compute the number and orientation of TMR dimers per ring as ADP and inorganic phosphate (9). In the absence of SP, the re- a function of the average number of TMR molecules per ring. The lease of ADP is the rate-limiting step in the catalytic cycle (10). TMR dimers thus serve as quantitative reporter of the allosteric state The addition of SP increases the rate of ATP consumption by of the system. The TMR dimers also serve as a surrogate for substrate GroEL by accelerating ADP/ATP exchange. A single SP binds to protein, substituting in a more homogeneous, quantifiable manner the apical domains of two or more GroEL subunits introducing a for the heterogeneous intersubunit, intraring, noncovalent cross- load on the ring in the form of noncovalent intersubunit cross- links provided by the substrate protein. The characteristic stimulation links that stabilize the T state. This constraint shifts the allosteric of the ATPase activity by substrate protein is also mimicked by the equilibrium in favor of the T state, whose weak affinity for nu- TMR dimers. Using an expanded version of the nested cooperativity cleotide leads to the dissociation of the product, ADP, effec- model, we determine values for the free energy of the TT to TR and tively increasing the rate of ATP consumption (11). TR to RR allosteric equilibria to be 27 ± 11 and 46 ± 2 kJ/mol, re- spectively. The free energy of unstacking of the TMR dimers was SP Binding Problem. Many GroEL SPs contain multiple GroES-like estimated at 2.6 ± 1.0 kJ/mol dimer. These results demonstrate that motifs of the generic sequence P-HHH-P-H, where P and H rep- GroEL can perform work during the T to R transition, supporting the resent polar and hydrophobic residues, respectively (12–14). The iterative annealing model of chaperonin function. association of these motifs with GroEL is difficult to quantitatively analyze due to three levels of heterogeneity (Fig. 1). First, for BIOPHYSICS AND allostery | substrate protein binding problem every one native SP, there exist multiple misfolded states that form an ensemble of structures, all of which have the potential to COMPUTATIONAL BIOLOGY he GroEL/GroES nano-machine facilitates the folding of interact with GroEL differently. The second level of heterogeneity Ta large number of diverse substrate proteins (SPs), converting is topological. Any one of the misfolded states has several GroEL them from one of many misfolded states to unique native states, binding motifs, which can be arranged in multiple ways between which are no longer recognized by GroEL. This machine is ul- the seven SP binding sites of a ring. The third level occurs within the timately powered by the binding and hydrolysis of ATP, driving binding pocket itself. Although the seven SP binding pockets of GroEL through a series of allosteric states. Two views of GroEL GroEL are all identical, they bind the general sequence P-HHH-P-H, function have emerged: one as a passive antiaggregation cham- which, being degenerate, covers a wide range of structural sequen- ber and the other as an active folding device, performing work on ces. Thus, within a single SP–GroEL complex, the various SP SP (1, 2). Our experiments support the latter view, demon- strating that GroEL has the ability to perform work on its SPs via Significance domain movements that accompany the allosteric transitions. Noncovalently “stacked” tetramethylrhodamine dimers are Allostery. The chaperonin protein GroEL consists of 14 57-kDa used to report and perturb the allosteric equilibrium in GroEL. subunits, arranged in two back-to-back heptameric rings (3). A The spectroscopic differences between the TMR monomers and GroEL subunit contains three distinct domains: apical, interme- dimers allow for quantitative measurements of the population diate, and equatorial domains. The equatorial domain houses the of allosteric states. The noncovalent intersubunit stacking in- nucleotide-binding pocket, whereas the site for SP and GroES teraction within a dimer mimics the cross-linking constraint binding is found between helices H and I in the apical domain (4). that SP places on the structure of GroEL. This feature allows The SP binding sites, primarily composed of hydrophobic residues, TMR dimers to be used as SP surrogates to quantitate the impact ’ line the inner wall of the heptameric ring s central cavity and bind of SP on the chaperonin cycle of GroEL. GroEL overcomes a load “ the exposed hydrophobic regions of the misfolded SP. The mo- of 7.8 kJ/mol, demonstrating its ability to perform work on SP. bile loops” of GroES displace the SP from this site (5, 6). In the chaperonin cycle, SP first binds to, and then becomes encapsu- Author contributions: N.C.C. and G.H.L. designed research; N.C.C. performed research; lated inside, GroEL’s barrel-like structure. After a short time (a N.C.C. and G.H.L. analyzed data; and N.C.C. and G.H.L. wrote the paper. few seconds at 37 °C), the SP is released, whether folded or not. The authors declare no conflict of interest. The chaperonin cycle involves a series of allosteric transitions Freely available online through the PNAS open access option. within a heptameric ring (7). In the absence of ATP, the ring See Commentary on page 10884. adopts the T state, which has a high affinity for SP, low affinity 1To whom correspondence should be addressed. E-mail: [email protected]. for nucleotide, and cannot bind GroES. The binding of ATP This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. triggersatransitiontotheR state, which is accompanied by a 1073/pnas.1307837110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1307837110 PNAS | Published online May 30, 2013 | E2451–E2459 Downloaded by guest on September 25, 2021 0 distribution of the surrogates across the subunits within a ring should be predictable. Finally, the surrogate should create an intersubunit load that is of uniform energetic value; i.e., the sta- bilization energy of each intersubunit cross-link should be iden- tical. These qualities can be found by using noncovalently stacked tetramethylrhodamine (TMR) dimers as an SP surrogate. I Tetramethylrhodamine. TMR is commonly used in biochemical studies due to its high absorbance and fluorescence in the visible region. It is often covalently attached using a thiol- or amine- reactive linker group. As in previous studies (15–17), we took ad- vantage 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). Fluorescence is forbidden from this state, and large decreases in fluorescence are consistently observed under conditions that favor dimer formation. II A blue shift in the peak absorbance indicates that the xanthene planes housing the electronic transition are oriented parallel to one another. Various studies have demonstrated that the xanthenes of the two TMR molecules are stacked in the dimer (16, 19, 20). TMR has been used as a spectroscopic probe in other bio- III molecular experiments due to the spectroscopic difference between thedimericandmonomericdyespecies.HereweexploitTMRas both an allosteric probe and an SP surrogate. The fluorophore can be covalently attached to the protein at a cysteine residue using maleimide chemistry. The self-association of TMR is distance de- Fig. 1. The substrate protein binding problem: a schematic illustration of the pendent, and dimers only form when two labeled residues are in heterogeneous nature of SP binding. Level 0: SP’snativeconfirmation. Level I: every SP has multiple misfolded states. Level II: each misfolded state can bind close proximity. To use TMR dimers as an allosteric probe, the to GroEL with different topological arrangements. Level III: each SP binding residues to which TMR is attached were chosen such that in the T contact will differ because GroEL binds the general sequence P-HHH-P-H. state they are close enough to allow dimer formation and in the R state they are distant enough to prevent dimer formation. binding site contacts differ from one another. This heterogeneity Results and Discussion fi ’ makes it dif cult to use GroEL s natural SPs to quantitatively TMR2-DTT as a Model System for Stacked TMR Dimers.
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