Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates
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Colleges, School and Institutes
- Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, India; Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, UK.
- Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
- Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, UK. Electronic address: firstname.lastname@example.org.
- Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, India. Electronic address: email@example.com.
The 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 interacts loosely with MalZ to allow the protein to change its conformation and fold while bound during the refolding process. These observations suggest that the minichaperone works by carrying out repeated cycles of binding aggregation-prone protein MalZ in a relatively compact conformation and in a partially folded but active state, and releasing them to attempt to fold in solution.
|Number of pages||11|
|Journal||Biochimica et Biophysica Acta|
|Early online date||2 Jun 2018|
|Publication status||Published - 1 Sep 2018|
- Protein folding, Minichaperone assisted protein folding, Folding intermediates, Maltodextrin glucosidase, Surface plasmon resonance