Methylene resonances: invaluable probes for the study of proteins – CH2PROBE

A fundamental understanding of biomolecular events requires knowledge of molecular structure at atomic resolution as well as a detailed description of time-dependent changes in molecular conformation. Indeed, internal motions occurring in biomolecules span a wide range of amplitudes and rates which critically assist them to perform biological functions. Nuclear Magnetic Resonance (NMR) represents a powerful tool to achieve structure/dynamics/function studies of proteins but it suffers from two major drawbacks which are its low sensitivity and the fast nuclear spin relaxation. This strongly limits both the size of the biomolecules that can be studied and the time window for NMR measurements. The CH2PROBE project aims at demonstrating that, as new molecular objects, glycine specifically-labelled proteins will push forward these two limitations. Using innovative NMR experiments, it will be shown that glycine residues offer accurate and unprecedented probes for the study of biological processes. We plan to demonstrate our new developments on the Pin1 protein, a peptidyl-prolyl cis-trans isomerase (PPIase) that plays a major role in various biological processes. Pin1 has already been the subject of intense experimental and computational research over the last two decades and numerous Pin1 ligands have been described. As such, Pin1 will serve as a benchmark for establishing the impact of our NMR developments, in the light of the published literature. Specifically, we will use the glycine residues of Pin1 to enhance the NMR sensitivity even for relatively high molecular weight systems (CH2-TROSY experiments), measure numerous scalar and dipolar couplings (Gly-s3NMR experiments), create long lived nuclear states (LLS) and reveal weakly populated conformations (CEST). These measurements, taken together with MD and DFT calculations, should provide us with a comprehensive view of the protein structure and dynamics. In addition, Pin1 will be studied in complex with ligands that will be designed to offer useful NMR probes and to impact both the protein structure and dynamics. Eventually, the NMR experiments developed in the CH2PROBE project will shed light into subtle conformational features in proteins and will establish the Glycine-NMR approach as a versatile tool for biomolecular studies.
These developments will be based on specifically-labelled protein samples, where all the amino acids contain 2H, 12C and 15N nuclei, except for the glycine residues. Since they are responsible for strong dipolar interactions, we will replace all the protons by deuterium in non-Gly residues, whereas 15N spin labels will be kept to allow standard NMR measurements. Such specifically-labelled proteins will be expressed by developing a novel cell-free approach. Once we will demonstrate the efficiency and the selectivity of the protein expression system, it will be possible to specifically incorporate any spin labels at the glycine positions. Indeed, glycine is commercially available with various combinations of NMR active spins, being furthermore the cheapest labelled amino acid. We will take full advantage of this opportunity to optimize the protein samples for each NMR applications. In addition, this new labelling route will allow us to propose innovative D-DNP approaches. For small molecules, signal enhancements by factors up to 50000 have be obtained using D-DNP. We will demonstrate that tremendous sensitivity improvement could be also obtained in proteins, which will pave the way for new applications in biomolecular NMR.
The CH2PROBE project will rely on a strong partnership between NMR spectroscopists (Partner 1, UPMC/ENS, Paris), biochemists (Partner 2, IBS, Grenoble), organic chemists (Partner 3, UCP, Cergy-P), and theoretical chemists (Partner 4, ILL, Lyon). Eventually, the developed methods could be applied to any proteins for which structural and dynamics data are lacking or are not accessible using conventional methods.