©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Mutant at Position 87 of the GroEL Chaperonin Is Affected in Protein Binding and ATP Hydrolysis (*)

Celeste Weiss , Pierre Goloubinoff (§)

From the (1) Department of Botany, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The highly conserved aspartic acid residue at position 87 of the Escherichia coli chaperonin GroEL was mutated to glutamic acid. When expressed in an E. coligroEL mutant strain deficient for phage morphogenesis, plasmid-encoded GroEL mutant D87E restored T phage morphogenesis. It did not, however, reactivate the transcription of a recombinant luciferase operon from Vibrio fischeri. In vitro, GroEL mutant D87E was found to be impaired in the ability to bind nonnative proteins and to hydrolyze ATP, resulting in less efficient refolding of urea-denatured ribulose-1,5-bisphosphate carboxylase/oxygenase. Mutant oligomer D87E GroEL was able to bind GroES as efficiently as wild-type GroEL. The conserved aspartic acid residue at position 87 located in the equatorial domain of GroEL (Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, A., Horwich, A. L., and Sigler, P. B.(1994) Nature 371, 578-586) is thus inferred to have a dual effect on the binding of nonnative proteins to the GroEL core chaperonin and on ATP hydrolysis.


INTRODUCTION

The GroEL protein of Escherichia coli is a member of the sequence-related family of molecular chaperones termed chaperonins, which are found in bacteria, chloroplasts, and mitochondria, and is related to the TCP1 family in the cytosol of eukaryotes (see Hemmingsen et al.(1988) and Lewis et al.(1992); for a review, see Hendrick and Hartl(1993)). In the cell, GroEL and the co-chaperonin GroES facilitate the folding of proteins during synthesis and transport across membranes (Bochkareva et al., 1988; Kusukawa et al., 1989; Cheng et al., 1989; Frydman et al., 1994) and are involved in the protection of proteins from heat shock (Kusukawa and Yura, 1988; Martin et al., 1992; Horwich et al., 1993). In vitro, GroEL and GroES facilitate the folding of a wide array of proteins (Goloubinoff et al., 1989; Mendoza et al., 1991; Viitanen et al., 1992). The GroEL oligomer is composed of two stacked rings of seven identical 57.3-kDa subunits with a cylindrical shape (Hendrix, 1979; Hohn et al., 1979). GroES is a heptameric ring of 10-kDa subunits (Tilly et al., 1981; Chandrasekhar et al., 1986) that can bind one end (Saibil et al., 1991; Langer et al., 1992) or both ends (Azem et al., 1994b; Schmidt et al., 1994b; Llorca et al., 1994) of the GroEL cylinder.

The GroEL oligomer can recognize and spontaneously bind nonnative proteins (Goloubinoff et al., 1989). The release of bound proteins in a folding-competent state requires the hydrolysis of ATP and, under stringent conditions, the presence of the co-chaperonin GroES (Goloubinoff et al., 1989; Schmidt et al., 1994a). The binding of ATP to GroEL and its subsequent hydrolysis occur with a high degree of cooperativity (Gray and Fersht, 1991) and drive a conformational change in the chaperonin molecule (Saibil et al., 1993) that results in the release of the folding protein (Martin et al., 1991). Mutant analysis, in conjunction with x-ray structure analysis of GroEL (Fenton et al., 1994; Braig et al., 1994), has provided a basis for understanding the role of individual residues in specific chaperonin functions such as ATP hydrolysis, binding of nonnative proteins, and binding of GroES. Thus, it was inferred from two severe charge mutations that the aspartic acid residue at position 87 of GroEL is located in the ATP-binding pocket. Similarly, a cluster of GroES-binding and protein-binding double mutants was identified in the apical domain of GroEL, facing the central cavity of the GroEL cylinder. This area was therefore inferred to constitute a part of the protein-binding site of the chaperonin (Fenton et al., 1994).

In this article, we mutated the highly conserved aspartic acid residue at position 87 of E. coli GroEL (Fig. 1) to glutamic acid. We found that this mutation primarily affects the binding of nonnative proteins to GroEL. It also affects ATP binding and hydrolysis, but not the binding of GroES.


Figure 1: Evolutionary variability among chaperonins. The amino acid sequence of GroEL from E. coli was aligned in region 70-110 with 42 GroEL, cpn60, and hsp60 sequences from the SwissProt Data Base (1993) using the Pileup algorithm (gap penalty of 3) in the 1991 Genetics Computer Group software package (Devereux et al., 1984). Below the E. coli sequence are listed the alternative amino acids found at each position in at least one of the aligned sequences. Underlined residues are also conserved in chaperonins TCP1 and TF55 from archaebacteria (Trent et al., 1991).




MATERIALS AND METHODS

Plasmid pTrcESL

The groEL gene was amplified by the polymerase chain reaction from plasmid pKT200 (Bloom et al., 1986) using DNA oligomers 5`-TGGTCGACAAAGACGTAAAATTCGGTA-3` and 5`-CCAAGCTTCTCGAGCTGGACGCACTCGC-3` that included the unique restriction sites SalI and HindIII at the 3`- and 5`-ends of the groEL gene, respectively. GroES was amplified from plasmid pKT200 using the DNA oligomers 5`-GACCATGGAATTCCGTCCATTGCATGATCGC-3` and 5`-GCGTCGACCATTATCTTTATTCCTTA-3`. The amplified DNA fragments containing groES and groEL genes were introduced into the multiple cloning site of pTrc99A (Pharmacia Biotech Inc.) and called pTrcESL. The double mutant that resulted from the introduction of a SalI restriction site at the N-terminal end of GroEL (thus introducing the A to V and A to D mutations at positions 2 and 3 of GroEL, respectively) did not exhibit different in vivo behavior from the wild-type GroEL protein (Hemmingsen et al. 1988).

Mutagenesis

Site-directed mutagenesis was carried out using the polymerase chain reaction (Higuchi, 1990) with pTrcESL as a DNA template and the following two DNA oligomers: 5`-GCTGCAGGCGAGGGTACCACC-3` and 5`-GGTGGTACCCTCGCCTGCAGC-3`. The resulting SalI-ClaI fragment, containing the D87E mutation in GroEL, was introduced into pTrcESL, and the resulting plasmid was called pTrc87. DNA sequencing of the entire groEL gene confirmed the D87E mutation.

Expression and Purification of GroEL and GroES

Upon induction of the trc promoter of pTrc87 with isopropyl-l-thio--D-galactopyranoside (40 µg/ml), mutant D87E GroEL was overexpressed to a level at least 50-fold higher than that of background GroEL levels of the E. coli host. Purification of GroEL was as described by Azem et al. (1994a) and that of GroES as described by Todd et al.(1993).

Electron Microscopy

Samples were applied to a glow-discharged, carbon-coated, collodion-covered 300-mesh copper grid and negatively stained with 1% aqueous uranyl acetate. Specimens were viewed with a Philips CM12 electron microscope operating at 100 kV. Micrographs were recorded on Kodak SO-163 emulsion at a nominal magnification of 75,000.

ATPase Activity

The hydrolysis of ATP by GroEL was measured as described by Azem et al. (1994a). GroEL (3 µM protomer) was incubated in 50 mM triethanolamine, pH 7.5, 10 mM KCl, 10 mM MgAc, and various concentrations of [-P]ATP (0.01-5 mM with a specific activity of 3 µCi/mmol) for 5 min at 37 °C. Unhydrolyzed ATP was separated from the P-labeled inorganic phosphate by adsorption on 5% activated charcoal in 20 mM HPO according to Bais(1975). The data for the velocity of the ATPase reaction were fitted to the Hill equation using the nonlinear regression method of the Enzfitter computerized program according to Leatherbarrow(1987). The following constants were derived (see ): K = V/[GroEL], K` = [S], and n = Hill coefficient.

Chemical Cross-linking of Chaperonin Oligomers

The binding of GroES to wild-type and mutant GroEL in solution was measured using chemical cross-linking at 37 °C with glutaraldehyde (0.22%) and SDS-polyacrylamide gel electrophoresis as described by Azem et al. (1994a, 1994b).

Refolding of Rubisco

In vitro chaperonin-dependent refolding of Rubisco from Rhodospirillum rubrum was assayed at 25 °C as described by Goloubinoff et al.(1989). Rubisco (20 µM) was denatured in 4 M urea and 10 mM dithiothreitol and then diluted 80-fold into a solution of 50 mM Tris, pH 7.5, 20 mM MgAc, 20 mM KCl, 1 mM dithiothreitol, 20 mM glucose, 1 mM ATP, 6 µM GroES (protomer), and 3 µM wild-type or mutant GroEL. Hexokinase (Sigma) was added to a final concentration of 40 µg/ml to stop the refolding reaction at the indicated time points. Rubisco activity was determined as described by Goloubinoff et al.(1989).

Inhibition of Protein Refolding

Pig heart mitochondrial malate dehydrogenase (9 µM monomer; Boehringer Mannheim), denatured in 4.5 M urea for 3 h at 25 °C, was diluted 70-fold into a refolding solution containing increasing amounts of mutant or wild-type GroEL, 10 mM KCl, 20 mM MgAc, 50 mM triethanolamine, pH 7.5, and 2 mM dithiothreitol. Malate dehydrogenase activity was assayed at 25 °C in 150 mM K phosphate buffer, pH 7.5, 0.5 mM oxalacetate, 2 mM dithiothreitol, and 0.2 mg/ml NADH by measuring the time-dependent change in the absorption of monochromatic light at 340 nm (Miller et al., 1993).

Genetic Complementation of Phage Morphogenesis

The E. coli mutant strain T carries a chromosomal mutation in the groEL gene that prevents maturation of -bacteriophage proheads (Takano and Kakefuda, 1972). T was transformed with either pTrcESL or pTrc87, and phage morphogenesis was tested using a ``spot test'' for plaque formation with T bacteriophage according to Revel(1980) and Greener et al.(1993).

Genetic Complementation of Luciferase Transcription

In E. coli, the transcription of a plasmid-encoded luminescence (lux) operon from Vibrio fischeri, pChV1 (Ulitzur and Kuhn, 1988), requires high levels of GroEL and GroES (Adar et al., 1992). Wild-type E. coli cells carrying pChV1 emit levels of visible light 10 times higher than T cells carrying pChV1 (Adar et al., 1992). Cell lawns of T carrying the compatible plasmid pTrcESL or pTrc87 in addition to pChV1 were visually tested in the dark for emission of light after a 20-h incubation at 30 °C.


RESULTS

Structure and Stability of Mutant D87E

To analyze the effect of the mutation on the structure and stability of the chaperonin oligomer, purified D87E GroEL molecules were observed by negative stain electron microscopy and compared with wild-type GroEL (Fig. 2). Mutant D87E oligomers are seen to be organized into characteristic tetradecameric double-layered cylinders (Hendrix, 1979; Hohn et al., 1979), which are indistinguishable from wild-type GroEL. D87E displayed the same mobility as wild-type GroEL on nondenaturing 7% polyacrylamide gels and was as stable as wild-type GroEL in the presence of 1 M urea (data not shown). However, at variance with the wild type, mutant GroEL was half dissociated into monomers in the presence of 2.25 M urea (data not shown), suggesting that under extreme conditions, the mutant is more prone to dissociation than the wild-type oligomer. Finally, cross-linking with glutaraldehyde as described by Azem et al. (1994a) confirmed that within the time scale of the experiments described, >95% of the mutant chaperonin was in the tetradecameric form (data not shown). Thus, the general structure of the chaperonin was not affected by the mutation.


Figure 2: Electron micrographs of mutant D87E and wild-type GroEL.



Genetic Complementation with Wild-type and Mutant GroEL

In E. coli, expression of plasmid-encoded (pChV1) luciferase operon from V. fischeri is under the transcriptional control of GroE chaperonins (Adar et al., 1992). Thus, wild-type E. coli cells harboring plasmid pChV1 emit 10-fold more visible light in the early stationary phase than T GroEL mutant cells. When E. coli T mutant cells harboring the two compatible plasmids pChV1 and pTrc99A were spotted with T bacteriophages, a plaque did not develop (Fig. 3A, cell lawn 2) and the cell lawn emitted very low levels of light (Fig. 3B, celllawn2). When, however, the GroE-less pTrc99A plasmid was replaced by pTrcESL, the T cell lawn emitted high levels of light (Fig. 3B, cell lawn 1) and developed a plaque (Fig. 3A, celllawn1). When pTrcESL was replaced by pTrc87 containing GroEL with the D87E mutation, an intermediate phenotype was observed: the cell lawn emitted only low levels of light (Fig. 3B, cell lawn 3) but successfully developed a T plaque (Fig. 3A, celllawn3). Thus, the mutant chaperonin is partially functional in vivo.


Figure 3: Genetic complementation of a GroEL mutant host strain. The E. coli GroEL mutant strain T (Takano and Kakefuda, 1972) was transformed with a plasmid, pChV1, containing the luciferase operon from V. fischeri (Ulitzur and Kuhn, 1988) and with a compatible plasmid: wild-type pTrcESL (celllawn1), GroE-less control plasmid pTrc99A (celllawn2), or mutant pTrc87 (celllawn3). Bacterial lawns on LB agar plates were spotted with 3 µl of T bacteriophage (10 plaque-forming units/ml). After a 20-h incubation at 30 °C, the bacterial lawn was visualized for the formation of a bacteriophage plaque in the light (A) and in the dark (B).



Chaperonin-assisted Refolding of Rubisco in Vitro

When compared with wild-type GroEL, the chaperonin-dependent refolding of the Rubisco enzyme by mutant D87E exhibited a slower rate and reached a maximum recovery of 25% that obtained with the wild-type chaperonin (Fig. 4).


Figure 4: Time course of Rubisco refolding by wild-type and mutant D87E GroEL. Rubisco (20 µM), denatured in 4 M urea, was diluted 80-fold into a refolding mixture containing 6 µM GroES, 1 mM ATP, and 3 µM wild-type () or mutant D87E () GroEL as described under ``Materials and Methods.'' Aliquots were removed at various time intervals, and Rubisco activity was determined as described by Goloubinoff et al. (1989). The percent recovery is calculated relative to the activity of the same concentration of native Rubisco.



Inhibition of Spontaneous Protein Refolding

At 25 °C, urea-denatured malate dehydrogenase can refold spontaneously into native enzyme (Miller et al., 1993). In the presence of GroEL, however, the malate dehydrogenase folding intermediates bind to the chaperonin and are prevented from refolding into an active enzyme. Fig. 5shows that the concentration of D87E GroEL required to inhibit half the spontaneous refolding of malate dehydrogenase is four times higher than for wild-type GroEL. This indicates that the spontaneous passive binding of nonnative protein is deficient in the mutant.


Figure 5: Inhibition of spontaneous malate dehydrogenase refolding by wild-type and mutant D87E GroEL. Malate dehydrogenase (MDH; 9 µM) in 4.5 M urea was diluted into a refolding solution containing increasing concentrations of wild-type () or mutant D87E () GroEL. Malate dehydrogenase activity was measured as described under ``Materials and Methods'' after 135 min of refolding at 25 °C.



Stability of the Binary Complex

When wild-type GroEL was first challenged with nonnative Rubisco, incubated with ATP for increasing amounts of time, and then supplemented with GroES to initiate refolding, no significant loss of recovered Rubisco was observed (Fig. 6, ). In contrast, when Rubisco-bound mutant D87E GroEL was incubated with ATP for increasing amounts of time and then supplemented with GroES, a significant time-dependent loss of recoverable Rubisco was observed (Fig. 6, ). If, however, the initial incubation was carried out in the absence of ATP, no loss of recoverable Rubisco activity was observed, either for wild-type or mutant D87E GroEL (Fig. 6, and , respectively). These results further indicate that the binding and rebinding of nonnative protein to the chaperonin are affected by the D87E mutation.


Figure 6: Rubisco recovery after incubation with or without ATP. Urea-denatured Rubisco was diluted as described in the legend of Fig. 4 in the presence of wild-type ( and ) or mutant D87E ( and ) GroEL. Binary complexes were incubated for increasing amounts of time in the presence ( and ) or absence ( and ) of ATP prior to the addition of GroES (6 µM). The GroES-dependent refolding reaction was allowed to progress for 15 min and then was arrested with glucose and hexokinase as in the legend of Fig. 4. The percent recovery is calculated from the Rubisco activity recovered when ATP and GroES were provided at the time of the Rubisco dilution.



ATPase Activity

The rate of ATP hydrolysis by mutant D87E is half that of wild-type GroEL (Fig. 7). summarizes the kinetic parameters for the ATPase activity of mutant and wild-type GroEL. The affinity of ATP for mutant D87E was nearly an order of magnitude lower than for wild-type GroEL. Moreover, ATP was hydrolyzed by the mutant in a noncooperative manner. However, GroES inhibited ATP hydrolysis similarly in mutant and wild-type GroEL (). This suggests that mutant D87E is deficient in the ability to bind and hydrolyze ATP, but not in the ability to bind GroES.


Figure 7: Time-dependent hydrolysis of ATP by wild-type () and mutant () GroEL.



Binding of GroESto Mutant D87E and Wild-type GroEL

The binding of GroES to mutant D87E or wild-type GroEL oligomers in the presence of ATP was measured using chemical cross-linking with glutaraldehyde and SDS-polyacrylamide gel electrophoresis as described by Azem et al. (1994b). In the presence of a saturating concentration of ATP, GroES has the same affinity for wild-type GroEL (Fig. 8A) as for mutant D87E GroEL (Fig. 8B). However, in the presence of a saturating concentration of GroES, GroES binding requires 5.7-fold less ATP to bind wild-type GroEL (Fig. 8C) compared with mutant D87E (Fig. 8D). The effective concentrations of ATP necessary for the binding of GroES to half of the GroEL oligomers were 34 and 6 µM for mutant and wild-type GroEL, respectively. This suggests that functional binding of GroES to GroEL is not directly affected by the D87E mutation, but rather through the decreased affinity of the mutant for ATP.


Figure 8: Binding of GroES to wild-type and mutant GroEL. Wild-type GroEL (3 µM protomer) (A) or D87E GroEL (B) was incubated with 1 mM ATP and increasing concentrations of GroES. The ratio between GroES and GroEL protomers was 0, 0.125, 0.25, 0.50, 0.75, 1.0, and 2.0 in lanes 1-7, respectively. Wild-type GroEL (3 µM) (C) or D87E GroEL (D) was incubated with an excess of GroES (6 µM) and increasing concentrations of ATP (0, 1, 3, 5, 10, 50, 100, and 500 µM ATP in lanes 1-8, respectively). Cross-linking of chaperonin hetero-oligomers with 0.22% glutaraldehyde, SDS-polyacrylamide gel electrophoresis, and Coomassie Blue staining was according to Azem et al. (1994a).




DISCUSSION

In this work, the highly conserved residue Asp-87 of GroEL chaperonins was mutated to glutamic acid. In a GroEL-deficient E. coli host cell, plasmid-encoded mutant D87E restored phage morphogenesis as efficiently as wild-type GroEL, but not the expression of the lux operon, indicating that the mutant chaperonin is partially functional in vivo. While under extreme conditions, the D87E mutant may be less stable in vitro than wild-type GroEL, it nevertheless assembled into a functional oligomer with the same apparent structure as wild-type GroEL. Moreover, mutant chaperonin D87E retained, albeit at various levels, all the chaperonin functions, thereby allowing an analysis of the relationship between ATP hydrolysis, GroES binding, protein binding, and protein folding in the oligomer.

The spontaneous formation of the chaperonin-malate dehydrogenase binary complex was four times less efficient for mutant D87E than for wild-type GroEL, indicating that mutant D87E has a lower affinity for nonnative proteins. Consistent with the 4-fold reduction in the ability of the mutant chaperonin to bind nonnative malate dehydrogenase, the maximal recovery of Rubisco by the mutant, in the presence of ATP and GroES, was 25% that of the wild-type chaperonin (Fig. 4).

The binary complex between mutant chaperonin and Rubisco was also less stable than the wild-type binary complex when incubated for increasing periods of times with ATP. Martin et al.(1991) suggested that ATP hydrolysis, in the absence of GroES, can cause bound proteins to undergo multiple futile cycles of release and rebinding to GroEL. While the wild-type binary complex appeared to withstand multiple ATPase-driven cycles of protein release and rebinding without a significant loss of recoverable Rubisco, the mutant binary complex was significantly destabilized by ATP hydrolysis (Fig. 5), suggesting that the rebinding of Rubisco to the mutant is less successful than to the wild-type molecule. This demonstrates that, although the mutation is in the ATP-binding pocket, the ATP hydrolysis mechanism is functional and is capable of driving reversible conformational changes in the mutant chaperonin. Furthermore, it confirms that mutant D87E is primarily affected in its ability to initially bind and to subsequently rebind nonnative proteins during ATP-driven cycles.

A kinetic analysis of ATP dependence curves of the GroEL ATPase showed that the maximal ATPase activity of the mutant was 50% that of the wild type and that the affinity of ATP for mutant D87E was an order of magnitude lower than for wild-type GroEL. Fenton et al.(1994) showed that two different charge mutations at the same position, D87K and D87N, resulted in a complete loss of ATPase activity and, consequently, of the protein refolding activity and GroES binding ability. The effects of our D87E mutation as well as of D87K and D87N are compatible with the structure analysis from x-ray crystallography, which places Asp-87 in the ATP-binding pocket of GroEL (Braig et al., 1994; Fenton et al., 1994; Kim et al., 1994). The fact that a significant level of protein refolding was achieved by a mutant lacking cooperativity in ATP hydrolysis suggests that cooperativity of ATP hydrolysis may not be an absolute requirement of the chaperonin-assisted protein folding mechanism, as previously suggested (Bochkareva et al., 1992; Jackson et al., 1993; Langer et al., 1992).

Higher concentrations of ATP were required for the binding of GroES to the mutant compared with the wild-type chaperonin oligomer (Fig. 8, C and D). This can be explained by the lower affinity of mutant D87E for ATP. A titration of the GroES-dependent binding of GroES to GroEL in the presence of a saturating concentration of ATP showed that GroES has the same affinity for wild-type GroEL as for mutant D87E GroEL (Fig. 8, A and B). This was confirmed independently when the same concentration of GroES inhibited half of the ATPase activity in the wild-type oligomer compared with the mutant oligomer (). Furthermore, the binding of GroES to the mutant was functional since it ultimately resulted in the refolding of Rubisco.

A cluster of GroEL mutants, all impaired in the ability to bind nonnative proteins, was shown to be located in the apical domain of GroEL, facing the upper central cavity of the oligomer (Braig et al., 1994; Fenton et al., 1994). Remarkably, all the protein-binding mutants were also found to be impaired in the ability to interact with GroES, suggesting that nonnative proteins and GroES compete for common sites in the inner face of the apical domain of the GroEL core oligomer (Fenton et al., 1994). Our findings suggest that the protein-binding sites are not necessarily located in the apical region of the central cavity, but may be located on the external envelope of the GroEL cylinder, in the equatorial domain of GroEL (Braig et al., 1994). This finding is of particular importance in view of recent observations that symmetric GroEL(GroES) oligomers, in which both access ways to the central cavity are obstructed, are nevertheless fully functional chaperonins capable of successfully assisting the refolding of nonnative proteins (Azem et al., 1994b).

  
Table: Characteristics of ATPase activity of wild-type and mutant D87E GroEL

The K` and n values for wild-type and mutant D87E GroEL were the average of eight and six experiments, respectively. Values for the inhibition by GroES of the GroEL ATPase were the average of four experiments. K values are from a representative experiment in which the purified mutant D87E protein was devoid of detectable degradation products as judged by native SDS gels (data not shown).



FOOTNOTES

*
This work was supported in part by Grant 00015/1 from the United States-Israel Binational Science Foundation, Grant 1180 from the Joint German-Israeli Research Program (to P. G.), and Grant 512 from the Levi Eshkol Fund (to C. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-26-585391; Fax: 972-26-584425; E-mail: pierre@huji.vms.ac.il.

The abbreviation used is: Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase.


ACKNOWLEDGEMENTS

We thank Martin Kessel for providing the electron micrograph in Fig. 2, S. Ulitzur for plasmid pChV1, G. Lorimer for purified Rubisco, and S. Diamant and A. Azem for critical review and discussions.


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