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1 Department of Biochemistry, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA
2 Département de Biochimie Médicale, University of Geneva, 1211 Geneva, Switzerland
3 Max-Planck-Institut für Biochemie, Department of Cellular Biochemistry, D-82152 Martinsried, Germany
Reprint requests to: Samuel J. Landry, Department of Biochemistry SL43, 1430 Tulane Avenue, New Orleans, LA 70112, USA; e-mail: landry{at}tulane.edu; fax: (504) 584-2739.
(RECEIVED March 26, 2004; FINAL REVISION May 4, 2004; ACCEPTED May 4, 2004)
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Abstract |
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-hairpins formed by the mobile loops restores the normal rate of dissociation. Thus, the free energy of mobile-loop ordering and disordering acts like the inertia of an engine’s flywheel by modulating the speed of chaperonin conformational changes. Keywords: nuclear magnetic resonance; surface plasmon resonance; ATP hydrolysis; folding funnel; allele-specific genetic interaction; intrinsically unstructured protein
Supplemental material: see www.proteinscience.org
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04773204.
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Introduction |
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The Escherichia coli chaperonin GroEL and its co-chaperonin GroES constitute a molecular machine that assists the folding of nascent and misfolded polypeptides. GroEL is composed of 14 identical 57-kDa subunits, which are organized into two heptameric rings that are stacked back to back, thus forming a double-ring structure (Braig et al. 1994). Each GroEL subunit has an equatorial domain that has ATPase activity and forms the interface between the two rings, an apical domain that binds client proteins and GroES (Fenton et al. 1994) and an intermediate domain that connects the equatorial and apical domains. GroES forms a single-ring structure consisting of seven identical 10-kDa subunits (Hunt et al. 1996). Each GroES subunit uses a mobile loop with a conserved hydrophobic tripeptide for interaction with GroEL (Landry et al. 1993). The mobile loops are ~16 amino acids in length and undergo a transition from disordered loops to -hairpins concomitant with binding the apical domains of GroEL (Shewmaker et al. 2001).
The GroE machine has been described picturesquely as a two-cylinder reciprocating engine (Lorimer 1997). Each ring of GroEL is negatively cooperative to the opposite ring (Yifrach and Horovitz 1995). The cooperative binding of ATP and GroES to the trans ring facilitates the departure of ADP and GroES from the cis ring (Todd et al. 1994). However, cycling time depends largely on the rate and cooperativity of ATP hydrolysis in the cis ring (Fridmann et al. 2002). The client protein binds to the inner surface of one of the GroEL rings (Braig et al. 1993). The subsequent binding of ATP and GroES to the client-bound GroEL ring causes the apical domains of that ring to reorient and sequester the hydrophobic surfaces used for binding the client protein (Xu et al. 1997). The client protein is released into the "Anfinsen cage" and potentially achieves a native fold (Saibil et al. 1993). The machine is believed to assist protein folding by preventing aggregation (Buchner et al. 1991), limiting the client protein’s conformational choices (Coyle et al. 1999; Brinker et al. 2001), and/or unfolding misfolded client protein molecules (Shtilerman et al. 1999; Chen et al. 2001).
The timing of articulations and energy transfers in a molecular machine must be tuned to match physical constraints, such as the molecular dimensions, temperature, and function. There exists a stringent interdependence between various alleles of E. coli groEL, and E. coli groES or bacteriophage-encoded co-chaperonin genes. Scores of mutant alleles that block and/or restore a functional interaction have been identified (Ang et al. 2000). These allelic interactions have two important features: First, many mutations that block the functional interaction of the proteins affect residues that are outside of the binding interface, and second, most of the mutations can be grouped into one of two phenotypic classes.
Bacteriophages T4 and RB49 encode co-chaperonins (Gp31 and CocO, respectively) that are distantly related to E. coli GroES (Ang et al. 2001; Keppel et al. 2002) and are required for successful bacteriophage propagation. During a bacteriophage T4 infection of E. coli, Gp31 functions with the host GroEL in folding the T4 coat protein Gp23 (Andreadis and Black 1998). Presumably, the bacteriophage RB49-encoded CocO homolog operates in a similar capacity. Why GroES is insufficient for folding Gp23 is unknown; however, it has been postulated on structural grounds that Gp31 and CocO, in conjunction with GroEL, form a larger "Anfinsen cage" (Hunt et al. 1997).
In a previous study, Richardson and coworkers classified GroEL(E191G) as a "low-affinity" GroEL mutant because it was unable to bind Gp31 (Richardson et al. 1999). Binding was restored with a mutant co-chaperonin encoded by T4 mutant 31(L35I), which had been selected to propagate on E. coli mutant groEL(E191G). T4 31(L35I) could not propagate on groEL(A383T) until it acquired a second mutation, resulting in T4 31(L35I, T31A). We proposed that T4 31(L35I) was incompatible with groEL(A383T) because the protein complex was overstabilized and that T4 31(L35I, T31A) was compatible with groEL(A383T) because T31A compensates for L35I. The substitutions L35I and T31A are expected to stabilize and destabilize the GroEL-bound -hairpin formed by the Gp31 mobile loop, respectively.
Here, we used genetic and biochemical approaches to test two hypotheses, namely, (1) whether excessive co-chaperonin binding affinity can effectively block chaperonin function, and (2) whether destabilizing the mobile loop -hairpin can reverse the excessive affinity and restore a functional interaction with a "high-affinity" GroEL mutant.
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Isolation of high-affinity GroEL mutants |
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-hairpin (Shewmaker et al. 2001). The rationale for believing the selected GroEL mutants would exhibit an higher affinity for GroES is twofold: First, they would be able to restore some of the interaction strength disrupted by the GroES(G24D) substitution, and second, they would be able to block bacteriophage T4 and RB49 propagation because of an excessive affinity for Gp31 and CocO, respectively. Following preliminary tests, we concentrated our efforts on two putative high-affinity GroEL mutant proteins, GroEL(V174F) (Zeilstra-Ryalls et al. 1994) and GroEL(E178K) (this work). Because these suppressor mutants were isolated in the context of a groES mutant allele, it was of interest to determine whether the putative high-affinity GroEL mutations are detrimental for E. coli growth. Tests for bacteriophage P1 cotransduction and for overexpression toxicity indicated that neither the groEL(V174F) nor the groEL(E178K) allele is compatible with the wild-type groES+ allele (Supplemental Material). Thus, we conclude that the GroES–GroEL(V174F) and GroES–GroEL(E178K) combinations cannot sustain E. coli viability.
Various derivatives of T4 bacteriophage, differing only in gene 31, were plated on E. coli strains with various groEL backgrounds to determine if the putative high-affinity GroEL mutants were compatible with wild-type Gp31, high-affinity Gp31(L35I), low-affinity Gp31(T31A), or the double mutant Gp31(T31A,L35I) (Richardson et al. 1999). Only T4 31(T31A) propagated on a bacterial host encoding either GroEL(V174F) or GroEL(E178K) at any temperature (Table 1
). This behavior is the exact opposite to the phenotype observed for the low-affinity strain encoding GroEL(E191G), on which only T4 31(L35I) propagated (Table 1).
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).
We constructed a low-affinity RB49 co-chaperonin by incorporating a glycine-for-serine substitution (S28G) at the position homologous to the site of the T31A substitution in bacteriophage T4 Gp31. S28G is expected to strongly disfavor -sheet formation (Minor and Kim 1994). The mutation encoding S28G was first introduced in a plasmid-borne gene and then recombined into the RB49 bacteriophage genome. As anticipated, the phenotype of RB49 cocO(S28G) was very similar to that of T4 31(T31A) (Table 1). When the S28G and Q36R substitutions were combined to produce RB49 cocO(S28G,Q36R), as for T4 31(T31A, L35I), the phenotypes of the two substitutions canceled each other out (Table 1).
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Tryptophan fluorescence enhancement in WGp31–GroEL complexes |
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). Thus, fluorescence-binding assays using the various purified WGp31 proteins could explain the phenotypes of mutations that affect chap-eronin function.
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). The difference between ATP and ADP complexes cannot be attributed to differences in degree of complex formation. When the fluorescence enhancements were analyzed as a function of ADP concentration, the plots revealed highly cooperative transitions, and the signals for all four WGp31 proteins achieved >95% of their maximum values at 1 mM ADP. Thus, the difference in enhancement for ATP and ADP complexes must be intrinsic to the complexes formed.
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Delayed dissociation of high-affinity WGp31–GroEL complexes |
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). Dissociation rates for the various WGp31 proteins were almost indistinguishable from each other (Fig. 1A, inset) and equal to the rate expected if dissociation was limited by the rate of ATP hydrolysis (one turnover every 10 to 12 sec; Todd et al. 1994), which is consistent with the ability of all of the mutant T4 bacteriophage to propagate on wild-type E. coli (Table 1).
As expected, the low-affinity chaperonin GroEL(E191G) bound weakly to WGp31(T31A,L35I) and not at all to WGp31(T31A) (Fig. 1B), which is consistent with the failure of T4 31(T31A,L35I) and T4 31(T31A) to propagate on the E. coli groEL(E191G) mutant host (Table 1).
The binding kinetics of high-affinity GroEL(E178K) and GroEL(V174F) revealed a defect associated with excessive co-chaperonin affinity. The dissociation rates for the various WGp31 proteins differed greatly (Fig. 1C,D), whereas association rates were indistinguishable from each other (Fig. 1; data not shown). Only WGp31(T31A) dissociated from the high-affinity chaperonins with wild-type rates, which is consistent with the unique ability of T4 31(T31A) to propagate on E. coli mutant strains that express the high-affinity chaperonins (Table 1). Dissociation rates for the other WGp31 proteins were much slower. The rank order of dissociation rates was WGp31(T31A) > WGp31(T31A, L35I) > WGp31 > WGp31(L35I), with decrements of ~10-fold from one WGp31 protein to the next. This ranking is consistent with the hypothesis that the corresponding co-chaperonin mutations were selected on the basis of restoring optimum binding affinity; for example, T4 31(L35I) was selected in the presence of GroEL(E191G) because L35I strengthened Gp31 binding, and T4 31(L35I, T31A) was selected in the presence of GroEL(A383T) because T31A weakened Gp31(L35I) binding.
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Delayed dissociation of high-affinity GroES–GroEL complexes |
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). The mutants GroEL(V174F) and GroEL(E178K) dissociated with very slow rates, 2.5 (±1) x 10–4 sec–1 and 0.4 (±0.2) x 10–4 sec–1, respectively, which are ~100-fold slower than the dissociation rate for wild-type GroEL (Hayer-Hartl et al. 1995). GroEL(E191G), a low-affinity mutant, showed a dissociation rate that was similar to that of wild-type GroEL (Fig. 2). |
Slower cycling of high-affinity WGp31–GroEL complexes |
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). This is consistent with previous studies finding that GroES inhibits the steady-state ATPase rate of GroEL by 50% in similar conditions (Todd et al. 1993). At this "high" potassium ion concentration (5 mM), GroES binding to one ring of GroEL prevents nucleotide exchange in that ring until ATP hydrolysis is complete (Todd et al. 1994). A new cycle begins with binding of ATP and GroES to the trans ring and release of GroES and ADP from the cis ring. At a low potassium ion concentration (1 mM), GroES blocks ADP/ATP exchange in the trans ring and therefore completely inhibits chaperonin cycling. In the presence of WGp31 and 5 mM potassium ion, the ATPase rate for the low-affinity GroEL(E191G) was inhibited by <50%, consistent with weak binding of WGp31 to GroEL. In contrast, the ATPase rates for high-affinity GroEL(V174F) and GroEL(E178K) were inhibited by >50%, suggesting that the stronger binding of WGp31 delays the cycling of the high-affinity GroEL mutants, analogous to the delay in cycling caused by GroES at intermediate potassium ion concentrations.
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Nascent formation of a -hairpin in the mobile loop correlates with GroEL binding affinity
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-hairpin that resembles the conformation adopted by mobile-loop peptides bound to GroEL (Landry et al. 1993, 1996, 1997; Shewmaker et al. 2001). Thus, mutations could increase or decrease GroEL-binding affinity by increasing or decreasing the tendency to form the -hairpin. As noted previously, the L35I and T31A substitutions are expected to increase and decrease the tendency to form -sheet, respectively (Shewmaker et al. 2001). Nascent structures in the mobile loops of WGp31 and variants were compared by analysis of H
chemical shifts (
H) obtained from two-dimensional nuclear magnetic resonance (NMR) spectra. Polypeptide backbone resonances corresponding to the most dynamic region of the mobile loops, primarily in the N-terminal flank of the hairpin turn (Landry et al. 1997, 1999), were consistently observed in spectra for all of the variants.
The H for residues D27, E28, E29, and S33 were notably sensitive to the mobile-loop mutations (Fig. 4). In comparison to the H for wild-type WGp31, the H for all four residues in WGp31(L35I) moved downfield, whereas the H for these residues in WGp31(T31A) and WGp31(T31A,L35I) moved upfield. Downfield shifts indicate an increased tendency to adopt the conformation, and upfield shifts indicate a decreased tendency to adopt the conformation (Wishart et al. 1991). Thus, L35I favors the GroEL-bound -hairpin conformation, and T31A disfavors the GroEL-bound -hairpin conformation. The resulting tendency for the mobile loops to form the -hairpin conformation follows the rank order, WGp31(T31A) < WGp31(T31A,L35I) < WGp31 < WGp31(L35I), which matches the rank order of dissociation rates, from fastest to slowest.
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Discussion |
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). The amino-acid substitution in high-affinity GroEL(V174F) is likely to disrupt a hydrophobic cluster that stabilizes apical domains in the down conformation, therefore shifting the population of GroEL subunits toward the up conformation (Ang et al. 2000; Klein and Georgopoulos 2001). We propose a similar explanation for the high-affinity phenotype of GroEL(E178K) (Supplementary Fig. 3). When the apical domain is in the down conformation, E178 forms a salt bridge with R322. Disruption of the salt bridge would shift the population toward the up conformation.
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-hairpins, which further stabilizes the GroEL apical domains in the up conformation. The T31A substitution compensates by destabilizing the GroEL-bound -hairpins. The stabilizing effect of L35I is not as strong as the destabilizing effect of T31A. The upfield changes in H chemical shift due to T31A were larger than the downfield changes due to L35I, and H chemical shifts for the double mutant were upfield from those of wild-type Gp31. The changes in -hairpin stability correlate with expected changes in intrinsic -sheet propensity from host–guest studies (Minor and Kim 1994). L35I is expected to stabilize the conformation by 0.49 kcal mole–1 when the affected position is in the hydrophobic center of a -sheet, an environment that may be similar to the -hairpin strand that is buried against GroEL (Fig. 4C). T31A is expected to destabilize the conformation by 0.83 kcal mole–1 when the affected position is at the solvent-exposed edge of a -sheet, an environment that may be similar to the -hairpin strand opposite to the GroEL-bound strand. Thus, T31A overcompensates for L35I, and this is evident in the faster dissociation from high-affinity GroEL proteins of Gp31(L35I,T31A) compared with wild-type Gp31.
Excessive affinity causes the chaperonin machine to stall in a complex that forms in the presence of ATP but not ADP. ADP complexes exhibit generally lower fluorescence intensity than the corresponding ATP complexes (Supplementary Fig. 2), suggesting that the ADP complexes have different structures, possibly with fewer or less buried tryptophans. Moreover, in contrast to expectation, the ADP complexes with high-affinity GroEL proteins dissociate faster than the ADP complexes with wild-type GroEL (Supplementary Fig. 4). These results can be explained by a failure of the ADP complexes to achieve the conformational state that exhibits high-affinity behavior.
The difference between ATP and ADP complexes could be related to the number of mobile loops that are ordered in the complex. The crystallographic structure of the GroES–GroEL–ADP complex, including a mimic of the ATP 
-phosphate, aluminum fluoride (AlFx), appeared to be identical to the ADP complex without AlFx, despite the fact that the complex with AlFx was much more stable (Chaudhry et al. 2003). However, poor resolution of the mobile loops and symmetry averaging of the electron density may have obscured differences in the ordering of individual GroES mobile loops. It is possible that the stability of the complex depends on the number of tightly bound mobile loops. The complex formed in ATP may have a greater number of tightly bound mobile loops than the complex formed in ADP. Likewise, high-affinity GroEL may have a greater number of tightly bound mobile loops than wild-type GroEL has in the complexes formed in ATP. This model implies that fewer than seven tightly bound mobile loops are necessary to stabilize the wild-type complex. In experiments with GroEL containing mixtures of wild-type and mutant apical domains, as few as one or two wild-type apical domains were sufficient for stable binding of GroES (Farr et al. 2000).
Ordering of the mobile loops creates a mechanism for the co-chaperonin to promote transitions between the two predominant GroEL conformations, analogous to the action of a flywheel in a reciprocating engine (Fig. 5). On binding of ATP, formation of the -hairpin and binding of the mobile loops drive GroEL subunits into the up conformation. After ATP hydrolysis, dissociation and unfolding of the mobile loops drive GroEL subunits into the down conformation. As for any two-state protein-folding transition, the pathway for ordering and disordering the mobile loop can be described by a funnel in which there are no significant enthalpic barriers, and thus the kinetics of ordering and disordering are determined by only the time required to search conformational space (Papoian and Wolynes 2003). Mobile-loop ordering and disordering can provide a smooth and gradual driving force for GroEL conformational changes as well as co-chaperonin binding and dissociation.
The intrinsic kinetics of mobile-loop disordering are not likely to limit the rate of co-chaperonin dissociation. NMR studies indicated that the -hairpin forms and dissolves on a timescale of no longer than milliseconds (Shewmaker et al. 2001), whereas wild-type co-chaperonin dissociation occurs on a timescale of seconds.
The thermodynamics of the co-chaperonin mobile-loop order–disorder transition govern the kinetics of the GroEL conformational change. The fact that the effects of amino acid substitutions in Gp31 and GroEL are additive suggests that they modulate a single kinetic step. If all seven subunits participate in binding, the difference in dissociation rates for WGp31 and WGp31(T31A) corresponds to a reduction in the kinetic barrier to dissociation of 0.4 kcal mole–1 per subunit (Supplementary Fig. 5). Because a T-for-A substitution can destabilize a -sheet by more than twice this amount, destabilization of the GroEL-bound -hairpin by T31A can explain the restoration of normal dissociation rates and chaperonin function with high-affinity GroEL proteins.
The paradigm of the co-chaperonin mobile loop suggests that the ordering of binding domains could be widely used to sustain appropriate kinetics in macromolecular machines and other processes that involve reversible multivalent protein–protein interactions. Because domain ordering depends on the contribution of many intramolecular and intermolecular bonds, the kinetics are robust and can be finely tuned.
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Materials and methods |
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Protein preparations
Wild-type GroEL was expressed in E. coli B178 from a pBAD22 derivative containing the groE operon between the EcoRI and HindIII sites (kind gift of France Keppel, University of Geneva). Mutations causing single amino-acid substitutions were introduced by standard site-directed mutagenesis protocols. Mutant GroEL proteins were expressed in B178-derived strains that possessed identical chromosomal mutations, thus preventing the formation of mixed oligomers. Purification of GroEL and its mutants was as described previously (Shewmaker et al. 2001).
Expression of wild-type and mutant WGp31 proteins was performed in E. coli MC1009, and the purification was a modification of the protocol previously described (van der Vies et al. 1994). Three liters of preheated LB medium (50 µg/mL ampicillin) was inoculated with an overnight culture (1:20 dilution) and incubated at 37°C with constant shaking. At OD600 = 0.6, protein expression was induced by the addition of 0.02% l-arabinose. Cells were harvested after 5 h of induction and resuspended in 34 mL of 10 mM Tris-HCl (pH 7.7), 100 mM NaCl, 1 mM EDTA, and 1 mM -mercaptoethanol. After resuspension, 1 mL of Sigma Protease Inhibitor Cocktail (cat. no. P-8465) was added, and the cells were lysed by passage through a French Pressure Cell three times.
The cell lysate was diluted to four times its volume and centrifuged for 30 min at 23,000g to remove debris. The supernatant was adjusted to 5% w/v with streptomycin sulfate and slowly mixed for 15–20 min at 4°C. The precipitated nucleic acid was removed by centrifugation at 23,000g for 15 min. The supernatant was brought to 36% ammonium-sulfate saturation at 4°C by slow addition of 100% saturated ammonium sulfate while stirring.
The precipitated protein was recovered by centrifugation for 30 min at 23,000g and then resuspended in 25 mL of Buffer Q (20 mM Tris-HCl at pH 7.7, 1 mM EDTA, 1 mM -mercaptoethanol). The protein solution was dialyzed against 2 L of the same buffer for ~4 h at 4°C and then centrifuged for 10 min at 33,000g to remove precipitated contaminants. The protein was loaded on Q-Sepharose (Pharmacia) equilibrated with Buffer Q. The column was washed with one bed volume of Buffer Q, and then the protein was eluted with a gradient of 0–0.8 M NaCl in Buffer Q. The Gp31-containing fractions were dialyzed overnight against 3 L Buffer H (20 mM sodium phosphate at pH 7.4, 1 mM EDTA, 2 mM -mercapto-ethanol). The protein was loaded onto a hydroxy-apatite column equilibrated with Buffer H. The column was washed with one bed volume of Buffer H, and the protein was eluted with a 0.02–0.275 M sodium-phosphate gradient (pH 7.4, 1 mM EDTA, 2 mM -mercaptoethanol). The Gp31-containing fractions were pooled and stored in 70% saturated ammonium sulfate.
Fluorescence spectroscopy
All fluorescence experiments were performed on a Photon Technologies Quanta Master luminescence spectrometer with a gloved cuvette holder attached to a water bath for temperature maintenance. Excitation was at 293 nm, and emission was recorded at 334 nm. All kinetic experiments were performed under constant stirring in a total volume of at least 1.5 mL. Component concentrations for individual experiments are given in figure and table legends. The proteins used for all experiments were recovered by sedimentation and resuspension after storage under 70% saturated ammonium sulfate. Thus, small amounts of residual NH4+ ions (submillimolar) are part of the experimental conditions.
Steady-state ATPase activity
The GroEL proteins were separated from the bulk of the solution by using centrifugal filter columns (Microcon YM-50, Millipore; cat. no. 42415). The liberated inorganic phosphate from the hydrolysis of ATP was then quantitated using the Enzchek Phosphate Assay Kit (Molecular Probes; cat. no. E6646). In brief, this assay allows for the quantitation of inorganic phosphate produced from the hydrolysis of ATP. The assay is based on the enzyme purine nucleoside phosphorylase, which can convert the substrate 2-amino-6-mercapto-7-methylpurine riboside into ribose 1-phosphate and 2-amino-6-mercapto-7-methyl-purine. This enzymatic conversion causes a spectrophotometric shift in maximum absorbance to 360 nm for the enzymatic product from 330 nm for the substrate. The rates of inorganic phosphate production were determined at several chaperonin concentrations, first without and then with equal concentrations of co-chaperonin. The plots of ATPase rate versus chaperonin concentration were linear, suggesting that differences in ATPase rate were not due to differences in chaperonin assembly. After linear regression of these plots, the slopes were taken as the ATPase rate constants.
NMR spectroscopy
Samples were prepared in 550 µL of 50 mM potassium phosphate buffer (pH 6.0), and then 60 µL of D2O, 5 µL 20% (w/v) 3,3,3-(trimethylsilyl)propionate (TSP) in D2O, and 1 µL of 80 mM NaN3 were added. The final concentration of wild-type or variant WGp31 was 0.5 mM. All NMR spectra were recorded on a Bruker DRX 500 equipped with a 5-mm Bruker TXI probe. Proton chemical shifts were referenced to TSP (0 ppm). The NOESY experiment (homonuclear correlation via dipolar coupling) used a NOESY mixing time of 200 msec; and phase-sensitive data were acquired using the States-TPPI method (Jeener et al. 1979). The TOCSY experiment (homonuclear Hartman-Hahn transfer) used the MLEV17 sequence for mixing (60 msec) and two power levels for excitation and spin lock; phase-sensitive data were acquired using the States-TPPI method (Bax and Davis 1985). In both pulse sequences, water was suppressed by using the 3-9-19 pulse sequence with gradients (Piotto et al. 1992; Sklenar et al. 1993). Spectra of width 6000 Hz were acquired with 2048 points in t2 and 512 increments in t1 and then processed into a frequency-domain matrix of size 2048 x 2048.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Abstract
Introduction
