Biology subjects

Henot, F.

Publications and source records attributed to Henot, F..

3 recordsLinked to original sources

bioRxiv2025

Toward Site-Specific Characterization of Structural Perturbations on Glycosylated Fc Using NMR at Natural Abundance

Monoclonal antibodies (mAbs) are leading therapeutic agents due to their high specificity and limited side effects. Ensuring their structural integrity under stress and maintaining batch consistency require robust quality control. Methyl 2D NMR has emerged as a powerful tool to probe mAb structure at natural isotopic abundance, enabling spectral fingerprint comparisons across production batches to detect subtle structural changes. However, extracting atomic-level structural information requires assignment of methyl resonances to their amino acids. While such assignments are available for several antigen-binding fragments (Fabs), no comprehensive assignment has been reported for the crystallisable fragment (Fc). In this study, we present the methyl group assignment of the 50-kDa Fc fragment of an immunoglobulin G1 (IgG1) antibody. Using cell-free expression, strategic isotopic labelling, and high-quality 2D and 3D NMR experiments, we successfully assigned 94% of methyl resonances of a non-glycosylated Fc. Given that therapeutic mAbs are typically produced in Chinese Hamster Ovary (CHO) cells, we transferred this assignment to the methyl spectrum of a glycosylated Fc fragment obtained by the enzymatic cleavage of a CHO-produced mAb at natural abundance, achieving 83% assignment coverage. This assignment was then used to investigate the impact of methionine oxidation on Fc structure at atomic resolution using NMR. The methyl group assignment transforms 2D methyl NMR fingerprinting into a powerful tool for quality control. It enables the direct comparison of spectra acquired on mAbs produced at natural abundance, allowing the detection and localisation of chemical modifications and structural changes without the need for isotopic labelling. This approach offers a robust solution for monitoring the structural integrity of therapeutic antibodies throughout development and manufacturing.

biochemistry↗

bioRxiv2025

A Fast and efficient strategy for the NMR assignment of Fab methyl groups

Owing to their high specificity and therapeutic effectiveness, monoclonal antibodies (mAbs) have rapidly become one of the leading classes of biologic drugs used to treat critical illnesses. The antigen-binding fragment (Fab) of mAbs plays a key role in the antigen recognition, so its structural characterization is essential, as even a slight change to its Higher Order Structure (HOS) can impact the antibodys potency. Recently, 2D methyl NMR has been introduced as a powerful method to assess both the structure and integrity of therapeutic Fab fragments. However, the identification of methyl group resonances in NMR spectra remains rare since Fabs are large heterodimers of [~]50 kDa. Here, we present the methyl group assignment of an IgG1 Fab produced in a cell-free system with an optimal isotope labelling. We first assigned 99% of the alanine, isoleucine, leucine, methionine, and valine methyl groups of the therapeutic Fab targeting LAMP1 antigen. Building on this assignment, we propose a "divide and conquer" strategy that exploits sequence identities to rapidly assign methyl groups of other IgG1 Fabs. We demonstrate that the assignment of the Fabs constant region can easily be transferred from one IgG1 to another and that the variable part of a new Fab can be assigned using smaller uniformly 15N,13C-labelled constructs. We applied our strategy to ipilimumabs Fab and, using the assignment of ipilimumabs variable part and Fab anti-LAMP1s constant part, we could transfer the assignment of 89% of the methyl-containing amino acids to the entire ipilimumab Fab without having to produce deuterated samples. This assignment strategy can be generalised to any other IgG1 Fabs provided that their constant regions are identical and the strategy can be adapted to accommodate the expression levels of the different variable domains. This new method drastically facilitates the Fab assignment process, making it suitable for the pharmaceutical timeline.

biochemistry↗

bioRxiv2022

Visualizing the Transiently Populated Closed-State of Human HSP90 ATP Binding Domain

HSP90 are abundant molecular chaperones, assisting the folding of several hundred client proteins, including substrates involved in tumor growth or neurodegenerative diseases. A complex set of large ATP-driven structural changes occurs during HSP90 functional cycle. However, the existence of such structural rearrangements in apo HSP90 has remained unclear. Here, we identified a metastable excited state in the isolated HSP90 ATP binding domain. We used solution NMR and mutagenesis to characterize structures of both ground and excited states. We demonstrated that in solution the HSP90 ATP binding domain transiently samples a functionally relevant ATP-lid closed state, distant by more than 30 [A] from the ground state. NMR relaxation and molecular dynamics were combined to characterize the energy landscape corresponding to the transition between these interconverting states. The precise description of the dynamics and structures sampled by human HSP90 ATP binding domain is a paramount piece of information for the future design of new therapeutic ligands.

biophysics↗