Not merely a passive co-chaperone : dynamic remodeling of protein substrate by GroES alone and in concert with GroEL

• Stopped‐flow folding experiments of human carbonic anhydrase II (HCA II) monitored by ANS fluorescence showed formation of an early molten globule intermediate. Folding of HCA II both in the presence of GroEL alone or GroES alone led to a loss of ANS binding compared to that in the spontaneous refolding process, showing that GroES alone is capable to interact with the refolding protein and that the molten globule substrate seems to be brought into a more unfolded state by both chaperonins. Moreover, an additive effect of the reduction of ANS binding during the early refolding stages was observed in the presence of GroEL+GroES, suggesting a concerted additive decrease in formation of molten globule by the chaperonins. The interactions during folding (from 50 ms to 3 h) between HCA II and GroEL alone, GroES alone, GroEL/ES and GroEL/ES/ATP was monitored in more detail using five fluorescence (AEDANS) labeled HCA II mutants and steady‐state and stopped‐flow Trp‐AEDANS FRET measurements. We observed that GroEL stretches the protein substrate as an early event in the folding process, when compared to spontaneous folding. Interestingly, GroES alone can interact with the folding protein leading to remodelling of the structure of the molten globule intermediate. Furthermore, GroES exerts additive stretching effects of the protein substrate in concert with GroEL. However, in the absence of GroEL the action by GroES is transient and does not affect the reactivation kinetics or final yield and thereby GroES does not exhibit classical chaperone activity, which is likely the reason why the independent GroES activity on protein substrates has gone undiscovered for such a long time.

Nyckelord

Chaperone

FRET

protein folding

molten‐globule

and carbonic anhydrase

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