1. Severe Oxidative Stress Causes Inactivation of DnaK and Activation of the Redox- Regulated Chaperone Hsp33 
Jeannette Winter, Katrin Linke, Anna Jatzek, Ursula Jakob 
Molecular Cell 
Volume 17, Issue 3, Pages (February 2005)
DOI: /j.molcel

2. Figure 1
Hsp33 Confers Resistance to Oxidative Stress at Elevated Temperatures
(A) MC4100 wild-type (•) or hslO (Hsp33 gene) (○) deletion strains were grown at 30°C in LB medium until OD of 0.5 was reached. Then, cells were supplemented with 4 mM H2O2 and either kept at 30°C (inset) or shifted to 45°C. Aliquots were removed and the viable titer was determined.
(B–D) ΔrpoH (JW49) (∇),ΔrpoH overexpressing Hsp33 (JW44) (■), or ΔrpoH overexpressing DnaK/DnaJ/GrpE (JW53) (♢) were grown at 30°C in LB medium supplemented with 0.4% (w/v) arabinose and 0.25 mM IPTG for 4 hr. Then, each strain was diluted 1:20 into preheated LB medium at 45°C containing either 4 mM H2O2 (B), 6 mM HOCl (C), or 3 mM diamide ([C], inset) or into preheated LB medium (D) at 50°C (heat shock). Then, the viable titer was determined.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Hsp33 and DnaK/DnaJ/GrpE Complement Each Other In Vivo
Colored overlay of colloidal blue-stained 2D gels of aggregated proteins isolated from ([A] and [B]) ΔrpoH strains (shown in red) and ΔrpoH strains overexpressing Hsp33 (shown in green) or ([C] and [D]) ΔrpoH strains (shown in red) and ΔrpoH strains overexpressing DnaK/DnaJ/GrpE (shown in green) after ([A] and [C]) heat stress treatment (40 min, 45°C) or ([B] and [D]) oxidative heat stress treatment (40 min, 45°C, 4 mM H2O2). Yellow spots correspond to proteins that show identical aggregation in both strains. Spots that appear orange or red aggregate to a lesser extent in Hsp33 or DnaK/DnaJ/GrpE-overproducing strains as compared to ΔrpoH strains that do not overexpress heat shock proteins. (E) shows selected spots from (A)–(D).
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Oxidative Inactivation of DnaK Is Induced by Low ATP Levels and Heat Stress
(A) Reversible inactivation of nucleotide-depleted DnaK in vitro. Nucleotide-depleted DnaK (20 μM) was incubated at 43°C in the presence of 2 mM H2O2. To monitor the activity of DnaK, the influence of DnaK on the DnaK/DnaJ/GrpE-mediated refolding of chemically denatured luciferase was tested. After 15 min of oxidative inactivation, the incubation of DnaK was continued at nonstress temperatures (30°C) in the absence (○) or in the presence (•) of reducing conditions (2.4 μM TrxA, 70 nM TrxB, 160 μM NADPH). Similar results were obtained when 5 mM GSSG was used during the inactivation of DnaK and 14 mM GSH to reactivate DnaK.
(B) Drop in intracellular ATP level under oxidative stress conditions. ΔrpoH strains overexpressing DnaK/DnaJ/GrpE were grown at 30°C until exponential phase was reached. Then, the culture was diluted 1:2 into prewarmed media at 43°C in the absence (○) or presence (•) of 4 mM H2O2. After 20 min stress, catalase was added to quickly degrade H2O2, and the cultures were shifted to 30°C. Aliquots of the cells were taken, and the total amount of ATP was determined. Inset: MC4100 strains were grown at 30°C until OD of 0.5 was reached. Then, the culture was shifted to 43°C and supplemented with either 4 mM H2O2, 6 mM HOCl, or 3 mM diamide. After 15 min of stress treatment, aliquots were removed and the total amount of ATP was determined.
(C) Oxidative inactivation of DnaK in vitro locks the N terminus of DnaK in an unfolded state. 0.5 μM DnaK or DnaK-Cys15Ala mutant (inset) was unfolded at 43°C in the (a) absence (dotted line) or (b and c) presence of 5 mM GSSG. As indicated by the arrow, after 15 min of incubation, the refolding of DnaK was initiated by the addition of a mix of 0.5 mM ATP and 14 mM GSH to a and c or only 0.5 mM ATP to b (gray line). After a further 15 min of incubation, 14 mM GSH was added to b.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 Inactivation of DnaK Is Reversible In Vivo
(A) DnaK is unable to prevent luciferase aggregation during oxidative heat stress in vivo. ΔrpoH strains overexpressing either DnaK/DnaJ/GrpE (left panel) or DnaK/DnaJ/GrpE/ClpB (right panel) were grown at 30°C to exponential phase. Then, luciferase expression was induced for 30 min, after which de novo protein synthesis was stopped for 30 min by addition of 50 μg/ml tetracycline. The culture was diluted into prewarmed media at 43°C in the absence or presence of 4 mM H2O2. Aliquots were taken before the stress treatment as well as 8 min after heat or oxidative heat stress treatment. Then, cells were lysed and aggregated proteins were separated from the soluble supernatant by centrifugation. Western blot analysis using antibodies against luciferase was performed.
(B and C) ΔrpoH overexpressing either no chaperones (■), DnaK/DnaJ/GrpE (•), or DnaK/DnaJ/GrpE/ClpB (▾) were grown at 30°C to exponential phase, and luciferase expression was induced as described. The culture was diluted into prewarmed media at 43°C in the absence (B) or presence (C) of 4 mM H2O2. After 8 min of stress treatment, catalase was added and cultures were shifted to 30°C (recovery period). Aliquots were removed at the indicated time points, and the amount of active luciferase was determined by luminescence. Only the recovery period is shown.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Model for the Inactivation of DnaK and Activation of Hsp33 under Oxidative Heat Stress and Their Interplay upon Return to Nonstress Conditions
Under heat stress conditions, the DnaK system is active and binds to unfolding substrate proteins, which are refolded upon return to nonstress conditions. Hsp33 is inactive under these conditions. Under oxidative heat stress conditions, the cellular ATP level drops and the N-terminal ATPase domain of DnaK unfolds. This results in the inactivation of DnaK. In contrast, Hsp33 becomes activated by disulfide bond formation and now binds to unfolding substrate proteins. Upon return to nonstress conditions, cellular ATP levels and reducing conditions are restored. Hsp33 becomes reduced and bound substrates are transferred to the DnaK system for refolding.
Molecular Cell  , DOI: ( /j.molcel )