It is well understood that molecular chaperones play an essential role in the prevention of aggregation and also in the correct folding of newly synthesized polypeptides in vivo as well as in vitro. Among the variety of molecular chaperones identified in E.coli, GroEL/GroES is the mostly investigated chaperone system reported in the literature. The essential chaperonin GroEL, acting with its cochaperonin GroES, has the ability to bind and assist the folding of a wide variety of client proteins. This process requires a complex allosteric reaction cycle, driven by ATP binding and hydrolysis, which involves concerted movements of the domains of this large molecular machine. It has long been known that isolated apical domains of GroEL (known as “minichaperone”) also show some chaperone ability, but relatively little attention has been paid to the mechanism by which these much simpler chaperones act. Therefore, in order to probe the mechanism of minichaperone action in detail, efficiency in the folding of relatively larger aggregation prone multidomain proteins and the fate of a polypeptide during a minichaperone-mediated folding reaction remain to be identified. Here, using surface plasmon resonance measurements provide an estimate for the equilibrium dissociation constant KD for the MalZ-minichaperone complex of 0.21 ± 0.04 μM, significantly higher than for most GroEL clients, showing that minichaperone interacts loosely with MalZ during the refolding process. Noticeably, we also found that, upon addition of GroES as an interference trap, MalZ is released from minichaperone in a predominantly non-native conformation that can be trapped by mutant forms of GroEL that are capable of binding but not releasing substrate. Released polypeptide undergoes kinetic partitioning: a fraction completes folding while the remainder is rebound by other minichaperone molecules. Folding appears to occur in an all-or-none manner, since, our evidence by using intrinsic and extrinsic fluorescence spectroscopy, trypsin sensitivity, enzymatic activity and gel filtration chromatography suggest that the refolded MalZ has the same structure as the native MalZ, but that its structure when bound to minichaperone is different. In conclusion, our results indicate that the minichaperone works through a process of iterative annealing, with repeated cycles of binding and release of the client protein. By this mechanism, minichaperone may lower the effective concentration of free, unfolded intermediates and reduce off-pathway processes such as aggregation or misfolding. Since folding intermediates of MalZ could not fold by itself in solution and hence requires assistance to fold, the transient release and binding of folding intermediates with minichaperone proceeds until it reaches the correct folded form. These finding will be value for researcher as minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon. Support or Funding Information Neha jain acknowledges joint Council of Scientific and Industrial Research-University of Grant Commission, Govt. of India for doctoral fellowship award no. 20-6/2008/ 2009. The authors acknowledge Indian Institute of Technology Delhi and the University of Birmingham for infrastructural support. Neha jain gratefully acknowledges financial support from Boeheringer Ingelheim Fonds to facilitate my experiments (SPR and thermofluor) at the University of Birmingham, United Kingdom This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.