New Routes for the Solution NMR Investigations of Extra Large Biomolecular Assemblies

Introduction

The cell is a collection of dynamic molecular machines. NMR spectroscopy is the method of choice to observe, at atomic resolution, complex conformational changes, transient interactions, and dynamics of proteins.

Background

Introduction of methyl specific labelling technology has enabled solution NMR studies of protein assemblies of several hundred kDa. However, this strategy is mostly restricted to symmetrical and thermostable protein assemblies, precluding applications to large medically relevant biological complexes.

Challenges

The increases of linewidths and the high number of signal overlaps hamper the NMR spectra analysis of most hetero-oligomeric large assemblies studied at room temperature.

Project Objectives

In this project, we will develop two complementary concepts to significantly simplify both NMR spectra and corresponding site-specific analysis:

1. We will build a multi-site specific labelling method enabling observation of NMR signals only for the sites of interest in a large protein complex.
2. We will invent a combinatorial strategy to reduce the time required for site-specific identification of each individual NMR signal from a few months to a few hours.

Methodology

New 1H-frequencies edition schemes will be introduced to enhance significantly the NMR spectra’s resolution of very slow tumbling biological particles.

Validation

These groundbreaking NMR methods will be validated using therapeutic antibodies (150 kDa), ribosome (2.4 MDa) samples, and will be used directly to capture the mechanisms of ATP-fueled human chaperonin (1 MDa) in complex with the aggregation-prone form of Huntingtin.

Anticipated Outcomes

This project will provide new technological breakthroughs to push biological applications of NMR significantly beyond its current boundaries. We anticipate that the simplification of the NMR analysis resulting from the newly developed NMR routes will transform solution NMR spectroscopy into a very competitive method to study large medically relevant biomolecular assemblies and molecular machines, so far considered as untargetable.