We propose a new approach, integrating nuclear magnetic resonance, electron paramagnetic resonance, and molecular dynamics simulations to unravel motions of disordered proteins.
In addition to state-of-the-art high0field NMR, we will use high-resolution relaxometry to investigate protein motions.
We have studied by NMR the disordered region of the protein Artemis, alone and in complex with Ligase IV (in progress). We have also worked on `nother protein, assigned its spectrum and analyzed its interactions.
Analysis of paramagnetic relaxation data with molecular dynamics simulations has been initiated.
EPR experiments were planned and necessary protein constructs prepared.
Over the next 15 months, we will obtain paramagnetic NMR, EPR, SAXS and molecular dynamics data on the systems under study. This is the core of the project. We will then achieve the objective of all these data: the integration of nanometric motions into a coherent and high-resolution description.
We have published two articles. The core of the project corresponds to work currently being performed in the teams of the 4 partners.
The discovery of intrinsically disordered proteins and regions (IDPRs) challenges our understanding of the physical chemistry of biological mechanisms. IDPRs increase the reach of biomolecular systems to project far and engage in multiple interactions, moving efficiently over nanometer distances. Yet, we still miss methods to investigate the geometry and timescales of these nanometer motions with high resolution.
We will develop an integrative experimental and computational framework to characterize nanometer motions in IDPRs at atomic resolution, exploiting synergies between paramagnetic NMR, electron paramagnetic resonance (EPR) and molecular dynamics (MD) simulations. A series of methodological innovations will be pursued in each of these fields: (1) we will tackle a key limitation that currently prohibits the quantitative interpretation of NMR relaxation effects due to the interaction with electrons by quantifying them over a broad range of the most relevant magnetic fields with a unique sample shuttle apparatus combined with high magnetic fields. (2) We will reduce the current flaws in molecular dynamics force fields for IDPs by direct improvement of the force fields and selection of MD trajectories based on experimental constraints with a new protocol. (3) The complementary information provided by paramagnetic NMR and EPR will allow us to carry an original quantitative analysis with MD simulations leading to an unprecedented description of the kinetics and conformational pathways of nanometer motions in IDPs with atomic resolution.
This methodology will be developed on the IDPRs from key proteins in the non-homologous end joining (NHEJ) pathway, a process essential for the repair of DNA double-strand breaks and adaptive immunity: the long disordered region of the enzyme Artemis and the IDPRs of the scaffolding and regulation proteins XLF and XRCC4. The consortium brings together specialists in NMR methodology and instrumentation, IDP NMR, molecular dynamics simulations, protein EPR, and the biology of NHEJ. The NANO-DISPRO project will provide new tools to investigate the kinetic and thermodynamic principles that underlie the function of IDPRs.
Monsieur Fabien Ferrage (Laboratoire des biomolécules)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
LBM Laboratoire des biomolécules
IBS INSTITUT DE BIOLOGIE STRUCTURALE
BIP Bioénergétique et ingénierie des protéines
CNRS DR12 _CRCM Centre National de la Recherche Scientifique Délégation Provence et Corse _Centre de recherche en cancérologie de Marseille
Help of the ANR 617,254 euros
Beginning and duration of the scientific project: September 2018 - 48 Months