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1 Supplementary Material Supplementary Materials and Methods Structure Determination of SR1-GroES-ADP AlF x SR1-GroES-ADP AlF x was purified as described in Materials and Methods for the wild type GroEL-GroES complex. Crystals were grown in buffer D (100 mm HEPES, ph 7.5, 100 mm KCl, 12-15% PEG 3000, 4% 1,3-propanediol) at 5 mg/ml protein in 1-10 µl hanging drops. After soaking for 30 sec in buffer D supplemented to 20% PEG 3000 and 20% ethylene glycol, crystals were frozen in liquid nitrogen. Data were collected to 7.5 Å at the ALS beamline on an ADSC Quantum 210 CCD detector and were processed using MOSFLM and SCALA (CCP4, 1994). Data were collected from two non-overlapping regions of the same crystal because of high sensitivity to radiation damage. The unit cell dimensions were determined as: a = Å, b = Å, c = Å, in the space group P The structure was solved by molecular replacement using the cis GroEL-GroES from the structure of the GroEL-GroES-ADP AlF 3 complex as a search model. Rigid body minimization reduced the R-factor to 46%. Calculation of AlF x Dissociation Constant The dissociation constant for AlF x was calculated from the equation: K AlFx =[AlF x ]/((K app /K BeFx )-1), where K AlFx is the dissociation constant for aluminum fluoride, K app is the apparent K d for BeF x binding in the presence of 50 µm AlFx, K BeFx is the dissociation constant for BeF x in the absence of AlF x. Supplementary Reference Collaborative Computational Project, 4. (1994). The CCP4 Suite: Programs for Protein Crystallography. Acta Crystallographica D50,

2 Supplementary Figure Legends Supplementary Fig. 1. Refolding of MDH is supported by ADP AlF x. Porcine mitochondrial MDH, substituted with tryptophan at aa 199, was unfolded in 0.1 M glycine, ph 2.5, and diluted into a buffered solution with SR1 to form a binary complex (6 µm SR1, 4 µm MDH monomer). This was mixed 1:1 in a stopped-flow apparatus with either 12 µm GroES and 2 mm ATP (red trace) or 12 µm GroES, 10 mm ADP, and AlF x complex (blue trace) as described in Materials and Methods. Refolding was monitored by following the increase in intrinsic Trp fluorescence (ex: 295 nm, em: >320 nm) with time. Supplementary Fig. 2. Non-native forms of rhodanese bound to SR1 are not released from the cavity wall by addition of ADP and GroES. (A) The assay for rhodanese release is shown schematically. Binary complexes were formed between unfolded 35 S-labeled rhodanese, 0.5 µm final concentration, and 1 µm SR1 as described for unlabeled rhodanese (see Materials and Methods). SR1-rhodanese binary complexes were supplemented to 10 µm with D87K, a GroEL trap molecule capable of binding but not releasing non-native polypeptides. Nucleotide (nuc.), either 1 mm ATP or 5 mm ADP, and 22 µm GroES were added, and the samples were incubated for 30 min at 25 C. During this incubation, released non-native rhodanese would be captured by the excess D87K. The samples were then treated with 15 mm CDTA to facilitate dissociation of GroES bound to SR1 and simultaneously release rhodanese if it had left the cavity wall during the incubation. The fate of the [ 35 S]rhodanese (i.e., release vs. persistent association with SR1 vs. transfer to D87K) was then determined by gel filtration on a 2

3 Tosohaas G4000SW xl HPLC gel filtration column equilibrated with buffer A (Materials and Methods). (B) Elution profile of a typical experiment showing the elution positions of trap D87K, SR1, GroES, and native rhodanese. (C) Without added nucleotide, rhodanese was recovered primarily at SR1, indicating formation of stable binary complexes. (D) In the presence of GroES and ATP, refolding of rhodanese occurs following release from the cavity wall into the stable cis chamber. After CDTAstimulated dissociation of GroES from SR1, the labeled protein is recovered at the position of native rhodanese. (E) ADP in the presence of GroES does not release nonnative rhodanese from SR1, because the labeled protein does not transfer to D87K after CDTA-stimulated dissociation of GroES, but remains with SR1. Inset: Elution profile showing that fluorescent GroES is completely released from SR1 by treatment with CDTA. Supplementary Fig. 3. GroEL-GroES-ADP AlF x complexes remain able to support productive folding of rhodanese for at least an hour after the addition of AlF x. The experiment is shown schematically on the left. Binary complexes were formed between 2 µm SR261, a single-ring form of GroEL with a cysteine residue at position 261 in the apical polypeptide binding surface, and 1 µm 35 S-labeled rhodanese in 0.5 ml (see Materials and Methods). Cysteine disulfide bonds were used to crosslink the non-native rhodanese (four cysteine residues) to SR261. This was achieved by diluting the sample to 2.5 ml with buffer A (Materials and Methods) without DTT and gel filtration on a PD- 10 column (Amersham) equilibrated in the same DTT-free buffer. Non-crosslinked rhodanese was then removed by mixing 3.0 ml of effluent with 0.2 ml of reactive red 3

4 resin (Sigma) for 2 hr at 25 o C. The resin was removed by centrifugation, and the protein concentrated to 4 µm by ultrafiltration using a Centricon 30 (Amicon). The efficiency of removal of non-crosslinked rhodanese was assessed by SDS-PAGE and Phosphorimager analysis of reactive red treated (+) and untreated (-) samples (panel on right). The sample was supplemented to 4 µm GroES and 5 mm ADP, and after 30 minutes at 25 o C it was further supplemented to 30 mm KF and 3 mm KAl(SO 4 ) 2 and equilibrated an additional 30 minutes. Free GroES was removed by gel filtration on a Tosohaas G4000SW xl column in buffer A without DTT. (In a separate control experiment with 35 S-labeled GroES and SR261, it was found that only ~10% of the SR261-rhodanese complexes could be recovered with GroES bound to them under these oxidizing conditions.) The recovered SR261-[ 35 S]rhodanese-ADP AlF x -GroES complex was concentrated to ~2 µm. After a total of ~60 min in non-reducing conditions, the folding-active complex was treated with 1 mm DTT (lower panel, +DTT) to reduce the disulfide bonds and initiate rhodanese refolding by releasing it from the cavity walls. Rhodanese activity was determined after a 30 min incubation at 25 o C. As compared with a control refolding experiment using an aliquot of the initial binary complex kept under reducing conditions (1 mm DTT) and supplemented to 1 mm ATP and 4 µm GroES in the presence of DTT, 8.4% rhodanese activity was recovered. Thus, >80% of those oxidized rhodanese complexes with a bound GroES molecule (10% of the starting material) refolded rhodanese to native form even 60 min after AlF x activation. Without the addition of DTT, no activity was recovered (lower panel, -DTT). 4

5 Supplementary Fig. 4. TLS libration tensors (Winn et al., 2001) for the apical domain and GroES for a representative protomer from the seven-fold symmetric GroEL-GroES complex. The Cα trace of a GroEL-GroES protomer is represented as follows: GroES subunit is colored in cyan, apical domains are red, intermediate domains are green, and equatorial domains are blue. The principal axes of the libration (rotation) tensors for the cis apical domain and GroES are shown in black for the ADP AlF 3 structure and gray for the ADP structure. The axes are scaled relative to the mean-squared libration amplitude about each axis and reveal the highly anisotropic character of the domain rotations, which are smaller in the ADP AlF 3 state, indicating decreased mobility of GroES and the cis apical domains. The libration tensors for the other domains are essentially identical between the two structures and are not shown. Supplementary Fig. 5. Crystal packing of GroEL-GroES-ADP AlF 3 complexes in P and SR1-GroES-ADP AlF x complexes in P (A) GroES packs into the trans ring of a neighboring complex in the GroEL-GroES P lattice, while (B), SR1- GroES forms a sparsely contacted, relatively unconstrained lattice in P with extensive solvent volumes between molecules. GroES is depicted in cyan; apical, intermediate, and equatorial domains are shown in red, green, and blue, respectively. 5

6 Supplementary Table I. Refinement statistics for GroEL-GroES-ADP complex Resolution (Å) Reflections ( F >0σ) Number of protein atoms Number of metal ions 7 Number of ADP molecules 7 R factor (%) a 26.9 Free R factor (%) b 28.7 RMS deviation in bond lengths (Å) RMS deviation in bond angles ( ) Average B factor c 88.2 Ramachandran statistics Most favorable (%) 89.6 Allowed (%) 9.5 Generously allowed (%) 0.9 Disallowed (%) a R factor =Σ F o - F c /Σ F o, where F c is the calculated structure factor. b R free is as R factor but calculated for 2% of randomly chosen reflections that were omitted from the refinement. c Average isotropic B-factor derived from TLSANL (Howlin et al., 1993). 6

7 Supplementary Table II. Data collection and structure determination of SR1-GroES- ADP AlF x Complex Cross-rotation search Significance Search structure: Cis ring of GroEL with GroES Euler angles Highest peak Highest false peak Φ1= Φ2= Φ3= σ Translation search Fractional coordinates Significance Highest peak Highest false peak x= y= z= σ 0.16σ Spacegroup P Cell Dimensions (Å) a b c Resolution (Å) Unique reflections Average redundancy a 3.4 [3.3] Completeness (%) a 88.4 [78.2] I/σI a 4.3 [1.8] 7

8 R a,b sym 0.16 [0.57] Mosaicity ( ) 1.2 R factor c a The value for the highest resolution bin ( Å) is given in brackets. b R sym =Σ I h -<I h > /ΣI h where <I h > is the average over Friedel and symmetry equivalents. c R factor =Σ F o - F c /Σ F o where F c is the calculated structure factor. 8

9 500 GroEL-MDH, +GroES, ATP GroEL-MDH, +GroES, ADP, AlFx 490 counts x time (sec)

10

11 HS HS SR261 rhodanese oxidize S S S S HS HS HS HS reactive red chromatography 80% 20% reactive red chromatography S S S S HS HS HS HS noncrosslinked rhodanese +ES,ADP ADP ADP ADP ADP 10% 90% +AlF x gel filtration +DTT -DTT Percent recovery of rhodanese activity

12

13 A B

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