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Having observed the effect of the charge properties of chaperonin cavity on folding of DM-MBP, we wanted to know how charges influence the folding of other known substrate proteins. To this end we took advantage of the flexible C-terminal tail of GroEL which consists of 4 Gly-Gly-Met (GGM) repeats and ends with an additional Met residue. These [GGM]4M sequences extend from the equatorial domain into the GroEL cavity but are not

resolved in the crystal structure (Braig et al., 1994). It has been shown previously that GroEL/ES cavity size (changed by deletion or addition of GGM repeats) plays an important role in protein folding (Tang et al., 2006). For our study, we engineered a series of mutants where we systematically mutated [GGM]4M to [GGX]4X where X represents either a

hydrophobic, charged or neutral amino acid. This would change the net charge of the cavity without affecting the cavity volume significantly (Table 4). GroEL-∆C is the GroEL mutant with the sequence [GGM]4M deleted.

Table 4: (GGM)4M (GGX)4X Amino acid residues Mass (Daltons) Vanderwaal’s volume (Ǻ3 ) Cis-cavity volume (Ǻ3 ) % change as compared to GroEL-WT Hydrophobicity* Met (M)WT 131.19 124 161.133 0 1.9 ΔC - - 168.161 +4.36 -

Aliphatic side chain

Gly (G) 57.05 48 163.793 +1.65 -0.4

Ala (A) 71.09 67 163.128 +1.24 1.8

Ile (I) 113.16 124 161.133 0 4.5

Aromatic side chain

Tyr (Y) 163.18 141 160.538 -0.37 -1.3

Polar neutral side chain

Ser (S) 87.08 73 162.918 +1.11 -0.8

Acidic side chain

Asp (D) 115.09 91 162.288 +0.72 -3.5

Basic side chain

Lys (K) 128.17 137 160.748 -0.24 -3.9

Volume of cis-cavity without C-terminal tail is 175.000 Ǻ3 * (Kyte and Doolittle, 1982).

Mutant chaperonins were overexpressed and purified from bacterial strain BL21(DE3). We verified that purified GroEL mutants formed tetradecamers similar to GroEL-WT (Wild type) by size exclusion chromatography and blue native gels.

Next to verify if chaperonin mutants bind unfolded substrate protein with affinity similar to GroEL-WT, we performed rhodanese aggregation prevention assay. Bovine mitochondrial rhodanese is a monomeric protein (33 kDa) comprised of two domains which catalyzes the formation of thiocyanide from thiosulfate and cyanide. It is highly aggregation prone protein and it has been shown that the folding of rhodanese on import into yeast mitochondria or expression in E.coli cytosol is dependent on Hsp60/Hsp10 or GroEL/ES respectively (Ewalt et al., 1997; Rospert et al., 1996) .Upon dilution from the denaturant into buffer alone, rhodanese rapidly aggregates which is reflected by increase in the absorbance at 320 nm. When denatured rhodanese is diluted into buffer containing 2-fold excess of GroEL- WT, no aggregation is observed (Figure 5.39). GroEL tail mutants bound unfolded rhodanese with affinity similar to GroEL-WT except for the mutant GroEL-GGD (Figure 5.39a) which had a lower binding affinity for rhodanese as compared to GroEL-WT. No aggregation was observed when 4-fold excess of GroEL-GGD over denatured rhodanese (Rhodanese: GroEL- GGD :: 1 : 4) was used (Figure 5.39b).

Figure 5.39: Inhibition of Rhodanese Aggregation by GroEL Tail Mutants.

Unfolded Rhodanese (25 μM) in 6 M GuHCl was diluted 100-fold dilution in buffer A alone (Spontaneous reaction) or in the presence of (a) wild-type GroEL or GroEL tail mutants (0.5 μM each) as indicated and (b) 1 μM GroEL-GGD. Aggregation was monitored by following absorbance at 320 nm on spectrophotometer. Spontaneous experiment after 10 min was set to 1.

In the rhodanese refolding experiment described later, increased amount of GroEL- GGD mutant over denatured rhodanese (Rhodanese: GroEL-GGD :: 1 : 4) was used due to its low binding affinity with Rhodanese.

Next, we measured the rates of ATP hydrolysis for GroEL-WT and GroEL tail mutantsr GroEL tail mutants in the absence or presence of GroES using a coupled ATP regenerating enzyme system. Except for the mutant GroEL-GGD which exhibited drastic decrease in ATP hydrolysis rate in presence of GroES, all GroEL tail mutants showed ~50% decrease in ATPase rate in presence of GroES like GroEL-WT (Figure 5.40). However, the absolute rates of ATP hydrolysis varied significantly for the GroEL tail mutants. This suggests that GroEL C-terminal tail mutants affect ATP binding and/or hydrolysis by GroEL directly or indirectly.

Figure 5.40: ATPase Activity of GroEL Tail Mutants. ATP hydrolysis rate was measured by following decrease in absorbance at 340 nm in presence of either GroEL alone (black) or GroEL and GroES (Grey). ATPase rates of GroEL are indicated as number of ATP hydrolyzed per GroEL tetradecamer per minute. Standard deviation of three independent measurements are shown.

Having established that the GroEL tail mutants can bind substrate protein and GroES, we next investigated the refolding of various substrate proteins. The refolding rate of 33 kDa rhodanese was significantly reduced for the mutants GroEL-GGY and GroEL-GGI, while only the yield of rhodanese folding was drastically affected in presence of GroEL- GGG.Rhoadanese refolding rate and yield was not significantly affected in presence of other GroEL tail mutants with different net cavity charge (Figure 5.41).

Figure 5.41: Rhodanese Refolding in Presence of GroEL Tail Mutants. Refolding of rhodanese was performed in presence of GroEL tail mutants. 25 µM rhodanese was denatured in 6 M GuHCl and 100-fold diluted in buffer A with 0.5 µM GroEL. 1 µM GroEL-GGD was used in the refolding reaction due to low binding affinity of this mutant with rhodanese. GroES was present in 2- fold excess of the chaperonin. Refolding was initiated by addition of 5 mM ATP at 25°C. Enzyme activity was measured at different time points by taking absorbance at 460 nm. Standard deviation of three independent measurements are shown. Bars indicate rates and circle indicates yield of refolding.

It is possible that the increased hydrophobicity of the cavity as compared to GroEL- WT hinders rhodanese refolding.

Next, we performed refolding experiments using 42 kDa DM-MBP as the substrate protein. Interestingly, the refolding rate of DM-MBP in presence of GroEL-GGI and GroEL-

GGY was similar to that of GroEL-WT, while the refolding of this substrate was significantly decreased in presence of other GroEL tail mutants. These findings suggest that the smaller 33 kDa rhodanese is not affected by changes in the charge properties of the cavity, while the 42 kDa DM-MBP folding is sensitive to the electrostatic property of the cavity. Also the overall hydrophobicity similar to wild-type GroEL cavity is optimal for folding of DM-MBP (Figure 5.42).

Figure 5.42: DM-MBP Refolding in Presence of GroEL Tail Mutants.

DM-MBP refolding was performed in presence of GroEL tail mutants. 25 µM DM-MBP was denatured in 6 M GuHCl and 100-fold diluted in buffer A with 0.5 µM GroEL. GroES was present in 2-fold excess of chaperonin. Refolding was initiated by addition of 5 mM ATP at 25°C and followed by monitoring Trp fluorescence with excitation at 295 nm and emission at 345 nm. Standard deviation of three independent measurements are shown.

Notably, the chaperonin-assisted refolding rates of both rhodanese and DM-MBP show no direct correlation with the ATP hydrolysis rate of the various tail mutants, suggesting that the assisted refolding of these monomeric proteins is independent of the repeated cycles of GroES binding.

The third substrate that we investigated was methylenetetrahydrofolate reductase (MetF) which catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5- methyltetrahydrofolate during methionine biosynthesis (Hatch et al., 1961). It is purified from

E.coli as tetramer with identical subunits of 33 kDa each (Guenther et al., 1999). It is one of the class III substrates of GroEL (Brinker et al., 2001) which cannot fold without the assistance of GroEL/ES system. Interestingly, when we checked the refolding rate of MetF in presence of GroEL tail mutants, we observed ~5-fold increase in refolding rate of MetF in presence of GroEL-GGK (Figure 5.43).

Figure 5.43: MetF Refolding in Presence of GroEL Tail Mutants. MetF refolding was performed in presence of GroEL tail mutants. 25 µM MetF was denatured in 6 M GuHCl and 100-fold diluted in buffer A with 0.5 µM GroEL (GroEL-assisted refolding). GroES was present in 2-fold excess of chaperonin. Refolding was initiated by addition of 5 mM ATP at 25°C. Enzyme activity was measured at different time points by taking absorbance at 343 nm. Standard deviation of three independent measurements are shown. Bars indicate rates and circle indicates yield of refolding.

This clearly indicates that GroEL-GGK alters the cavity environment in a way that accelerates the formation of native state of MetF.

These findings strongly suggest that the net charge or the fine balance between the overall hydrophobic and hydrophilic character of the GroEL cavity is of profound significance to promote folding of certain substrate proteins. While the charge effects on specific protein may vary, the overall environment of the GroEL cavity is optimal for folding of diverse set of substrate proteins. Also, the ATPase rate seems to have no direct effect on the folding of substrate proteins tested, consistent with the fact that SR-EL which undergoes just one round of ATP hydrolysis can refold substrate with a similar rate and yield like GroEL-WT (Weissman et al., 1996).