Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates

Publication date: September 2018Source: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Volume 1866, Issue 9Author(s): Neha Jain, Timothy J. Knowles, Peter A. Lund, Tapan K. ChaudhuriAbstractThe isolated apical domain of GroEL consisting of residues 191–345 (known as “minichaperone”) binds and assists the folding of a wide variety of client proteins without GroES and ATP, but the mechanism of its action is still unknown. In order to probe into the matter, we have examined minichaperone-mediated folding of a large aggregation prone protein Maltodextrin-glucosidase (MalZ). The key objective was to identify whether MalZ exists free in solution, or remains bound to, or cycling on and off the minichaperone during the refolding process. When GroES was introduced during refolding process, production of the native MalZ was inhibited. We also observed the same findings with a trap mutant of GroEL, which stably captures a predominantly non-native MalZ released from minichaperone during refolding process, but does not release it. Tryptophan and ANS fluorescence measurements indicated that refolded MalZ has the same structure as the native MalZ, but that its structure when bound to minichaperone is different. Surface plasmon resonance measurements provide an estimate for the equilibrium dissociation constant KD for the MalZ-minichaperone complex of 0.21 ± 0.04 μM, which are significantly higher than for most GroEL clients. This showed that minichaperone inter...

Source: Biochimica et Biophysica Acta (BBA) Proteins and Proteomics - July 11, 2018 Category: Biochemistry Source Type: research