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

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Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates. / Jain, Neha; Knowles, Timothy J; Lund, Peter A; Chaudhuri, Tapan K.

In: Biochimica et Biophysica Acta, Vol. 1866, No. 9, 01.09.2018, p. 941-951.

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@article{371abbfbf05c4c5c83957dc530c70511,
title = "Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates",
abstract = "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.",
keywords = "Protein folding, Minichaperone assisted protein folding, Folding intermediates, Maltodextrin glucosidase, Surface plasmon resonance",
author = "Neha Jain and Knowles, {Timothy J} and Lund, {Peter A} and Chaudhuri, {Tapan K}",
note = "Copyright {\textcopyright} 2018. Published by Elsevier B.V.",
year = "2018",
month = sep,
day = "1",
doi = "10.1016/j.bbapap.2018.05.015",
language = "English",
volume = "1866",
pages = "941--951",
journal = "Biochimica et Biophysica Acta",
issn = "1874-9399",
publisher = "Elsevier",
number = "9",

}

RIS

TY - JOUR

T1 - Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates

AU - Jain, Neha

AU - Knowles, Timothy J

AU - Lund, Peter A

AU - Chaudhuri, Tapan K

N1 - Copyright © 2018. Published by Elsevier B.V.

PY - 2018/9/1

Y1 - 2018/9/1

N2 - 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.

AB - 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.

KW - Protein folding

KW - Minichaperone assisted protein folding

KW - Folding intermediates

KW - Maltodextrin glucosidase

KW - Surface plasmon resonance

U2 - 10.1016/j.bbapap.2018.05.015

DO - 10.1016/j.bbapap.2018.05.015

M3 - Article

C2 - 29864530

VL - 1866

SP - 941

EP - 951

JO - Biochimica et Biophysica Acta

JF - Biochimica et Biophysica Acta

SN - 1874-9399

IS - 9

ER -