TY - JOUR
T1 - A Single-Molecule Strategy to Capture Non-native Intramolecular and Intermolecular Protein Disulfide Bridges
AU - Mora, Marc
AU - Board, Stephanie
AU - Languin-Cattoën, Olivier
AU - Masino, Laura
AU - Stirnemann, Guillaume
AU - Garcia-Manyes, Sergi
N1 - Funding Information:
We thank Ainhoa Lezamiz, Dr. Palma Rico, and Dr. Elena Rostkova for protein expression and purification. M.M. was funded by a Fight for Sight PhD studentship. This work was supported in part by the Francis Crick Institute, which receives its core funding from Cancer Research U.K. (FC001002), the U.K Medical Research Council (FC001002), and the Wellcome Trust (FC001002). For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. This work was supported by the European Commission (Mechanocontrol, Grant Agreement 731957), BBSRC sLOLA (BB/V003518/1) Leverhulme Trust Research Leadership Award RL-2016-015, Wellcome Trust Investigator Award 212218/Z/18/Z, and Royal Society Wolfson Fellowship RSWF/R3/183006 all to S.G.-M.
Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.
PY - 2022/5/25
Y1 - 2022/5/25
N2 - Non-native disulfide bonds are dynamic covalent bridges that form post-translationally between two cysteines within the same protein (intramolecular) or with a neighboring protein (intermolecular), frequently due to changes in the cellular redox potential. The reversible formation of non-native disulfides is intimately linked to alterations in protein function; while they can provide a mechanism to protect against cysteine overoxidation, they are also involved in the early stages of protein multimerization, a hallmark of several protein aggregation diseases. Yet their identification using current protein chemistry technology remains challenging, mainly because of their fleeting reactivity. Here, we use single-molecule spectroscopy AFM and molecular dynamics simulations to capture both intra- and intermolecular disulfide bonds in γD-crystallin, a cysteine-rich, structural human lens protein involved in age-related eye cataracts. Our approach showcases the power of mechanical force as a conformational probe in dynamically evolving proteins and presents a platform to detect non-native disulfide bridges with single-molecule resolution.
AB - Non-native disulfide bonds are dynamic covalent bridges that form post-translationally between two cysteines within the same protein (intramolecular) or with a neighboring protein (intermolecular), frequently due to changes in the cellular redox potential. The reversible formation of non-native disulfides is intimately linked to alterations in protein function; while they can provide a mechanism to protect against cysteine overoxidation, they are also involved in the early stages of protein multimerization, a hallmark of several protein aggregation diseases. Yet their identification using current protein chemistry technology remains challenging, mainly because of their fleeting reactivity. Here, we use single-molecule spectroscopy AFM and molecular dynamics simulations to capture both intra- and intermolecular disulfide bonds in γD-crystallin, a cysteine-rich, structural human lens protein involved in age-related eye cataracts. Our approach showcases the power of mechanical force as a conformational probe in dynamically evolving proteins and presents a platform to detect non-native disulfide bridges with single-molecule resolution.
KW - atomic force microscopy (AFM)
KW - non-native disulfide bonds
KW - protein folding
KW - protein mechanochemistry
KW - protein nanomechanics
KW - single-molecule force spectroscopy
UR - http://www.scopus.com/inward/record.url?scp=85131017575&partnerID=8YFLogxK
U2 - 10.1021/acs.nanolett.2c00043
DO - 10.1021/acs.nanolett.2c00043
M3 - Article
C2 - 35549281
AN - SCOPUS:85131017575
SN - 1530-6984
VL - 22
SP - 3922
EP - 3930
JO - Nano Letters
JF - Nano Letters
IS - 10
ER -