Biology subjects

Charpentier, B.

Publications and source records attributed to Charpentier, B..

2 recordsLinked to original sources

bioRxiv2025

R2TP-like Quaternary Chaperones: a comprehensive overview to understand the dynamic R2SP complex

The human R2SP complex belongs to the R2TP-like quaternary chaperone family and consists of RUVBL1, RUVBL2, SPAG1 and PIH1D2. R2SP is crucial for the correct assembly of motile cilia (SPAG1 null mutations cause Primary Ciliary Dyskinesia) and the organization of the synaptic zone. RUVBL1/2 ATPases are the powerhouse of this molecular machinery, while SPAG1 and PIH1D2 would be adaptors that interact with specific clients to promote their quaternary assembly. Despite these functional data, little is known about the structure of R2SP and the precise mode of action of these R2TP-like complexes. We have combined biochemical and structural approaches (NMR, structural mass spectrometry and cryo-EM) to investigate the 3D organization of the human R2SP complex, its mode of assembly and ATPase activity. Our study reveals a three-dimensional structure similar to that of the canonical R2TP complex, but also highlights differences in the mode of action of its RUVBL1/2 ATPase core as well as the binding of its adaptors SPAG1 and PIH1D2.

biophysics↗

bioRxiv2023

Denaturing mass photometry for straightforward optimization of protein-protein cross-linking reactions at single-molecule level

Mass photometry (MP) is a versatile, fast and low sample-consuming biophysical technique that gained interest in structural biology to study noncovalent assemblies in native conditions. We report here on a novel method to perform MP analysis in denaturing conditions (dMP) and its application for fast, accurate and straightforward optimization of chemical reactions in cross-linking mass spectrometry (XL-MS) workflows. dMP consists in a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. The proposed single-molecule method clearly overcomes the limitations and outperforms gold standard SDS-PAGE, as illustrated on several biological complexes. dMP provides an unprecedented and unmatched in-solution quantification of all coexisting XL species, including sub-complexes and non-specific XL aggregates, along with identification of significantly higher numbers of XL dipeptides in MS. We anticipate single-molecule dMP to be a high-impact game-changer for the XL-MS community with the potential to leverage the quality and reliability of XL-MS datasets.

biophysics↗