A Single Ring Is Sufficient for Productive Chaperonin-Mediated Folding In Vivo  Kåre L. Nielsen, Nicholas J. Cowan  Molecular Cell  Volume 2, Issue 1, Pages 93-99 (July 1998) DOI: 10.1016/S1097-2765(00)80117-3

Figure 1 An Altered Form of Hsp60 Containing Mutations in the Equatorial Domain that Are Designed to Preclude Ring–Ring Interactions (Hsp60SR1) Has Undiminished Folding Activity Compared to Unaltered Hsp60 (A and B) Folding reactions done with mitochondrial MDH (A) and bacterial OTC (B). Native control shows the activity (arbitrarily taken as 1) of an amount of nondenatured MDH identical to that used in the in vitro folding assays. Molecular Cell 1998 2, 93-99DOI: (10.1016/S1097-2765(00)80117-3)

Figure 2 The ATPase Activities of GroEL and Hsp60 Are Inhibited to Different Extents upon Interaction with Their Cognate Cochaperonins Initial rates of ATP hydrolysis by GroEL (circles) and Hsp60 (triangles) in the presence (closed symbols) or absence (open symbols) of their cognate cochaperonins Hsp10 and GroES, present in 4-fold molar excess with respect to chaperonin. Molecular Cell 1998 2, 93-99DOI: (10.1016/S1097-2765(00)80117-3)

Figure 3 Construction and Properties of Chimeric Chaperonins (A) Assembly of constructs encoding chimeric chaperonins. Sequences encoding GroEL and Hsp60 are shown as solid and shaded lines, respectively; amino acid numbers (GroEL) correspond to junction points. Arrows denote primers (not shown to scale) used in PCR reactions to generate fragments used to assemble chimeric constructs. PCR-generated fragments are shown as encoding portions of GroEL (G) or Hsp60 (H), with superscripts denoting apical (Ap) or equatorial (Eq1: N-terminal segment; Eq2: C-terminal segment) domains. (B) Subunit structure of chimeric chaperonins. Each chaperonin consists of an apical domain (circles) joined by an intermediate domain (vertical elipses) to an equatorial domain (horizontal rectangles). Domains derived from Hsp60 and GroEL are shown unshaded and shaded, respectively. Vertical bars in equatorial domains depict altered residues in GroEL (or homologous residues in Hsp60; see text) that in GroEL prevent interaction between rings (Weissman et al. 1995). (C and D) Gel filtration (Superose 6, 1.0 × 30 cm) profiles (C) (with the elution volume of the major peak(s) shown in ml) and appearance in the electron microscope ([D], end and side views, upper and lower panels, respectively) of the chimeric chaperonins shown in (B). Molecular Cell 1998 2, 93-99DOI: (10.1016/S1097-2765(00)80117-3)

Figure 3 Construction and Properties of Chimeric Chaperonins (A) Assembly of constructs encoding chimeric chaperonins. Sequences encoding GroEL and Hsp60 are shown as solid and shaded lines, respectively; amino acid numbers (GroEL) correspond to junction points. Arrows denote primers (not shown to scale) used in PCR reactions to generate fragments used to assemble chimeric constructs. PCR-generated fragments are shown as encoding portions of GroEL (G) or Hsp60 (H), with superscripts denoting apical (Ap) or equatorial (Eq1: N-terminal segment; Eq2: C-terminal segment) domains. (B) Subunit structure of chimeric chaperonins. Each chaperonin consists of an apical domain (circles) joined by an intermediate domain (vertical elipses) to an equatorial domain (horizontal rectangles). Domains derived from Hsp60 and GroEL are shown unshaded and shaded, respectively. Vertical bars in equatorial domains depict altered residues in GroEL (or homologous residues in Hsp60; see text) that in GroEL prevent interaction between rings (Weissman et al. 1995). (C and D) Gel filtration (Superose 6, 1.0 × 30 cm) profiles (C) (with the elution volume of the major peak(s) shown in ml) and appearance in the electron microscope ([D], end and side views, upper and lower panels, respectively) of the chimeric chaperonins shown in (B). Molecular Cell 1998 2, 93-99DOI: (10.1016/S1097-2765(00)80117-3)

Figure 4 A Single Ring Is Sufficient for Productive Chaperonin-Mediated Folding (A and B) Folding activities of chimeric chaperonins using unfolded MDH (A) or OTC (B) as target protein either in the presence or absence of the cochaperonins GroES or Hsp10. Bars drawn with broken lines show the yield of folded product in experiments done using the chimeric chaperonin GroELwtA/Hsp60wtE with or without 50 μM GroES or Hsp10 at 44°C, as compared with wild-type GroEL or Hsp60 under these conditions. (C) Generation of native MDH in chaperonin-mediated folding reactions as measured by consumption of NADH following the onset of folding by addition of ATP. Molecular Cell 1998 2, 93-99DOI: (10.1016/S1097-2765(00)80117-3)

Figure 5 The Single-Ring Chimera Hsp60wtA/GroELSR1E Functions via Multiple Cycles of Binding and Release of Hsp10 (A) MDH folding reactions done using chaperonin with (+) or without (−) preincubation with ATP and cochaperonin (Hsp10 in the case of Hsp60wt, Hsp60SR1, and Hsp60wtA/GroELSR1E; GroES in the case of GroELSR1). (B) Target protein (MDH) was presented to chaperonin (150 nM, calculated as heptamer) in multiple successive equimolar additions (see Experimental Procedures) such that the ultimate stoichiometric ratio of MDH:chaperonin was 9:1, and the enzymatic activity measured. One unit equals the amount of enzyme activity that can be produced from one round of chaperonin-mediated folding in the reaction, determined by measuring the activity of 150 nM MDH (calculated as monomer). Molecular Cell 1998 2, 93-99DOI: (10.1016/S1097-2765(00)80117-3)

Figure 6 Single-Ring Chaperonins Support Facilitated Protein Folding In Vivo Constructs engineered for the expression of various chaperonins and cochaperonins were introduced into wild-type or mutant (GroEL44 or GroES619) strains of E. coli together with a plasmid (pBC-luxAB) engineered for the IPTG-inducible expression of bacterial luciferase (see Experimental Procedures) and the maximum light emission determined as relative light units (RLU) per second. Closed diamonds, wild type E.coli; open triangles, GroES619; open squares, GroEL44. (A), pBC-luxAB alone; (B), pGroE-Hsp60; (C), pGroE-GroEL; (D), pGroE-Hsp60-Hsp10; (E), pGroE-Hsp60wtA/GroELwtE-Hsp10; (F), pGroE-Hsp60wtA/GroELSR1E-Hsp10. Molecular Cell 1998 2, 93-99DOI: (10.1016/S1097-2765(00)80117-3)

Figure 7 A Vast Difference Exists in the Interaction between Chaperonins (GroEL and Hsp60) and Their Respective Cochaperonins (A–D) Interactions between GroEL and GroES (A and C) and Hsp60 and Hsp10 (B and D) in the presence of either ATP (A and B) or ADP (C and D). Inset shows Scatchard plot of the data in (C). Note that in (D), association of the labeled probe with its target was undetectable. (E) Model of Hsp60/Hsp10 function. Molecular Cell 1998 2, 93-99DOI: (10.1016/S1097-2765(00)80117-3)