Final Report Summary - 2F4BIODYN (Two-Field Nuclear Magnetic Resonance Spectroscopy for the Exploration of Biomolecular Dynamics)
Proteins are small molecular machines that perform a task: their function. How do proteins achieve this task? Precisely, how do they move to perform their function? Nuclear magnetic resonance (NMR) is a unique spectroscopic technique to explore matter and molecules at atomic scale. In particular, relaxation, the process of return to equilibrium of the little atomic magnets (nuclear spins) that NMR manipulates, is directly dependent on molecular motions. The frequencies at which relaxation probes motions is proportional to the magnetic field so, to probe motions at low frequencies, corresponding to nanosecond timescales, we need to lower the magnetic field way below the magnetic fields used by high-resolution NMR. In this project, we have championed approaches where high-field and low-field NMR are combined together in each experiment to explore dynamics in biomolecules.
In order to reach low magnetic fields on a high-field spectrometer, we need to move the sample quickly to chosen positions in the stray field. We have performed this by using a sample shuttle that moves the sample at speeds up to 10 m/s. With a sample shuttle, we have measured relaxation at magnetic fields two to three orders of magnitude smaller than the field of the high-field magnet but benefit from the high-resolution (atomic resolution) of high fields. We call this approach high-resolution relaxometry. We have developed NMR experiments, theory and software to record and analyze such relaxation experiments for the protein backbone or its side chains. We have determined the nature of the motions of side chains using molecular dynamics simulations. In proteins that do not fold in a well-defined structure, which we call intrinsically disordered proteins, we have determined the distribution of motions on picosecond to nanosecond timescales and how these distributions were altered by the interaction with a partner.
The analysis of high-resolution relaxometry relies on some hypotheses because we cannot control the evolution of nuclear spins when the sample leaves the high-field magnet. In order to validate the analysis tools we have developed, we have built a new type of spectrometer that couples two magnetic centers at vastly different fields. We call this a two-field NMR spectrometer. The two-field NMR spectrometer has been used to measure accurate site-specific relaxation rates at magnetic fields that are incompatible with high-field relaxation. These measurements have allowed us to refine and validate our analysis for both protein sidechains and for disordered proteins.
The two-field NMR spectrometer is a fantastic instrument. Reaching far beyond the measurement of relaxation, we have introduced a new field: two-field NMR spectroscopy; a type of NMR experiments where the parts that benefit from high fields are carried at high fields and the parts that benefit from low fields are carried at low field. This is a change of paradigm for NMR, as conventional NMR experiments are carried out at a single magnetic field that is, at best, a compromise. We have shown on a series of proofs of concept that two-field NMR allowed the development of many new experiments and, in particular, opened the way to the study of dynamic systems inaccessible to conventional NMR.
This project has set the ground for a new field in NMR for the exploration of many challenging dynamic biomolecules. The applications of the methods developed in the course of this project will lead to a better, more detailed, and more accurate understanding of the motions of biomolecules that underlie their functions.
In order to reach low magnetic fields on a high-field spectrometer, we need to move the sample quickly to chosen positions in the stray field. We have performed this by using a sample shuttle that moves the sample at speeds up to 10 m/s. With a sample shuttle, we have measured relaxation at magnetic fields two to three orders of magnitude smaller than the field of the high-field magnet but benefit from the high-resolution (atomic resolution) of high fields. We call this approach high-resolution relaxometry. We have developed NMR experiments, theory and software to record and analyze such relaxation experiments for the protein backbone or its side chains. We have determined the nature of the motions of side chains using molecular dynamics simulations. In proteins that do not fold in a well-defined structure, which we call intrinsically disordered proteins, we have determined the distribution of motions on picosecond to nanosecond timescales and how these distributions were altered by the interaction with a partner.
The analysis of high-resolution relaxometry relies on some hypotheses because we cannot control the evolution of nuclear spins when the sample leaves the high-field magnet. In order to validate the analysis tools we have developed, we have built a new type of spectrometer that couples two magnetic centers at vastly different fields. We call this a two-field NMR spectrometer. The two-field NMR spectrometer has been used to measure accurate site-specific relaxation rates at magnetic fields that are incompatible with high-field relaxation. These measurements have allowed us to refine and validate our analysis for both protein sidechains and for disordered proteins.
The two-field NMR spectrometer is a fantastic instrument. Reaching far beyond the measurement of relaxation, we have introduced a new field: two-field NMR spectroscopy; a type of NMR experiments where the parts that benefit from high fields are carried at high fields and the parts that benefit from low fields are carried at low field. This is a change of paradigm for NMR, as conventional NMR experiments are carried out at a single magnetic field that is, at best, a compromise. We have shown on a series of proofs of concept that two-field NMR allowed the development of many new experiments and, in particular, opened the way to the study of dynamic systems inaccessible to conventional NMR.
This project has set the ground for a new field in NMR for the exploration of many challenging dynamic biomolecules. The applications of the methods developed in the course of this project will lead to a better, more detailed, and more accurate understanding of the motions of biomolecules that underlie their functions.