Figure 5.

Minichaperone does not assist the folding of MBP5. Denatured MBP5 in 6 M guanidine-HCl was rapidly diluted (>50-fold) into buffer D containing the indicated chaperones. All reactions were carried out at 37°C and folding was monitored by changes in intrinsic tryptophan fluorescence intensity (excitation: 295 nm, emission: 344 nm). The fluorescence intensity is reported as the fraction relative to native MBP5. (A) The effect of maltose on stability of MBP5. Native MBP5 was produced by folding in the presence of GroEL, GroES, and ATP followed by purification on a gel filtration column. Purified MBP5 was then diluted into buffer D plus 4 M urea. Shown is the time course of denaturation, as monitored by UV fluorescence, in the presence or absence of 10 mM maltose. (B) Folding of MBP5 in the presence of various chaperones. The concentration of MBP5 in folding reactions was 60 nM. The following concentrations of chaperones were present in the reactions: 1.75 μM GroEL and 1.75 μM GroES protomer (EL/ES/ATP); 1.75 μM GroEL protomer (EL/ATP); 1.75 μM sht-GroEL_191–345 (sht345); 0 μM GroEL (Spont.). We also failed to detect any significant recovery of native MBP5 in the presence of a 5-fold higher or 5-fold lower concentrations of minichaperone (data not shown). 2.5 mM ATP was present, where indicated. (C) Rescue of an MBP5-sht345 folding reaction by GroEL-GroES. The course of the reaction is indicated schematically by the time line: At time zero, denatured MBP5 was diluted into buffer D containing 1.75 μM sht-GroEL_191–345. After 1,200 sec, 3.5 μM GroEL, 3.5 μM GroES and 2.5 mM ATP were added.