Periodic Reporting for period 2 - BIO-enMR (New routes for magnetic resonance spectroscopy of biomolecules by combining EPR and NMR methods)
Reporting period: 2023-05-01 to 2024-10-31
Magnetic resonance spectroscopy is one of the most important tools to achieve structural and functional information in chemistry, physics, biology and medicine. However, one severe limitation is the inherent low sensitivity, associated with the small energy splitting of nuclear spins at external magnetic fields produced by available technologies. Latest developments in this field are taking advantage of the larger (about three orders of magnitude) magnetic moment of paramagnetic molecules to enhance the sensitivity of nuclear magnetic resonance (NMR). However, most protocols suffer from lack of general applicability and difficult implementation to existing instruments.
This project aims at developing new experimental designs by combining concepts of NMR with electron spin or paramagnetic resonance spectroscopy (ESR/EPR), which detects instead the spin of atoms or paramagnetic molecules. Electron and nuclear spins in molecules are mutually coupled by magnetic interactions, the mechanism of which can be strategically used to cross-fertilize NMR and EPR, for instance by selecting suited spin labels with specific physical and chemical properties. Research in this direction is taking advantage from shared developments in radio frequencies and microwave technologies as well similar designs of magnetization transfer experiments for representative applications to biomolecular systems, otherwise not feasible.
It is anticipated that a successful establishment, combined with current progress in instrumentation, i.e. the age of magnetic resonance in the GHz (NMR) and THz (EPR) frequency range, will offer new opportunities for analytical and biophysical investigations. Examples are studies of the activation mechanism, subunit interactions and inhibition of enzymes such as ribonucleotide reductases, which are important targets of cancer drugs, or NMR investigations of enzyme interactions with drugs, which are currently hampered by low sensitivity and resolution.
This project aims at developing new experimental designs by combining concepts of NMR with electron spin or paramagnetic resonance spectroscopy (ESR/EPR), which detects instead the spin of atoms or paramagnetic molecules. Electron and nuclear spins in molecules are mutually coupled by magnetic interactions, the mechanism of which can be strategically used to cross-fertilize NMR and EPR, for instance by selecting suited spin labels with specific physical and chemical properties. Research in this direction is taking advantage from shared developments in radio frequencies and microwave technologies as well similar designs of magnetization transfer experiments for representative applications to biomolecular systems, otherwise not feasible.
It is anticipated that a successful establishment, combined with current progress in instrumentation, i.e. the age of magnetic resonance in the GHz (NMR) and THz (EPR) frequency range, will offer new opportunities for analytical and biophysical investigations. Examples are studies of the activation mechanism, subunit interactions and inhibition of enzymes such as ribonucleotide reductases, which are important targets of cancer drugs, or NMR investigations of enzyme interactions with drugs, which are currently hampered by low sensitivity and resolution.
We are developing spectroscopic methods to detect nuclear spins with enhanced sensitivity using two different approaches. In one case, we detect nuclear magnetic moments via the larger magnetic moment of a paramagnetic molecule, a method called electron-nuclear double resonance (ENDOR). Specifically, we have introduced ENDOR detection of 19F or 17O nuclei to obtain structural information in biomolecules. The dipolar coupling of 19F with a paramagnetic spin label turned out to be sensitive to intermolecular distances in the angstrom to nanometer scale (≲ 2 nm), allowing investigation of active site structures in proteins and RNAs with high sensitivity and precision. For instance, in the enzyme ribonucleotide reductase, a 250 kDa macromolecular complex, we could use 19F ENDOR to measure the distance between two redox-active tyrosines across two protein subunits, shining light on the role of interface residues to controlling communication between these two subunits. These experiments would have not been feasible by direct 19F NMR detection, due to the much lower sensitivity. Also, 17O has offered unique opportunities to detect water molecules in the active RNR enzyme or to study the hydration structure of radical species involved in catalytic reactions.
In a second approach, we have implemented a new instrumental design for NMR signal enhancements using Overhauser dynamic nuclear polarization (DNP). This method relies on pumping with microwaves a paramagnetic molecule, called polarizer, which is mixed to the sample of interest. In analogy to an ENDOR experiment, magnetization is transferred between electron and nuclear spins. At an external magnetic field of 9.4 Tesla, the pumping frequency, to be on resonant with the electron Larmor frequency, requires sub-THz irradiation. The latter is a major bottleneck for liquids, where microwave penetration in the large NMR samples is strongly attenuated and leads to sample heating. In our work we could show a solution for these technical challenges. Empowered by a thin-layer setup in a cylindric NMR tube and microwave engineering, our design allows for hyperpolarized liquid-state NMR spectra in one and two dimensions with a reasonable sample volume (a few µL). We anticipate that our approach provides a starting point for a broader implementation of DNP signal enhancement in liquid-state NMR. The reached sensitivity opens up perspectives for structural determination of natural products or drugs, available in small quantities.
In a second approach, we have implemented a new instrumental design for NMR signal enhancements using Overhauser dynamic nuclear polarization (DNP). This method relies on pumping with microwaves a paramagnetic molecule, called polarizer, which is mixed to the sample of interest. In analogy to an ENDOR experiment, magnetization is transferred between electron and nuclear spins. At an external magnetic field of 9.4 Tesla, the pumping frequency, to be on resonant with the electron Larmor frequency, requires sub-THz irradiation. The latter is a major bottleneck for liquids, where microwave penetration in the large NMR samples is strongly attenuated and leads to sample heating. In our work we could show a solution for these technical challenges. Empowered by a thin-layer setup in a cylindric NMR tube and microwave engineering, our design allows for hyperpolarized liquid-state NMR spectra in one and two dimensions with a reasonable sample volume (a few µL). We anticipate that our approach provides a starting point for a broader implementation of DNP signal enhancement in liquid-state NMR. The reached sensitivity opens up perspectives for structural determination of natural products or drugs, available in small quantities.
During the first period of the project, the 19F ENDOR method has emerged as a general tool for molecular distance measurements, complementing the pool of methods for structural biology and refining molecular distance measurement with sub-angstrom precision. Still the open challenge is the interpretation of data in biological systems, which intrinsically contain contributions of complex conformational distributions. For this goal, experiments have to be conceived that possibly suppress other sources of inhomogeneous magnetic contributions in the ENDOR spectral line width, so that conformational distributions can be more easily deconvoluted. This will add to the understanding of conformational distributions that is key to understand structural and functional relationships. Our approach will be the design of new pulse sequences combined with new methods of spectral analysis, such as Bayesian inference of prior information from other sources, such as cryo-EM or X-ray structures or quantum chemical and molecular dynamic simulations.
On the second part of the project, after having demonstrated enhanced liquid-state NMR spectroscopy by Overhauser DNP, the focus will be on an improved instrumental design with no compromise in state-of-the-art NMR performance. We will examine the combination of our DNP probe design with cryogenic probes, which currently delivers highest NMR sensitivity. Other developments will include further increase of sample volumes (currently our volumes are still ≲ 10-fold smaller than in a standard NMR), temperature homogeneity and reduction of MW losses through materials in sample space, which would render the method compatible with low-cost MW sources. On the physical side, the Overhauser mechanism in many heteronuclei is still unexplored. Understanding these mechanistic details will be critical to the reach larger NMR signal enhancements. Nonetheless, we foresee that the simplicity of in-situ DNP will boost future research, in which the full potential of liquid NMR can be exploited.
On the second part of the project, after having demonstrated enhanced liquid-state NMR spectroscopy by Overhauser DNP, the focus will be on an improved instrumental design with no compromise in state-of-the-art NMR performance. We will examine the combination of our DNP probe design with cryogenic probes, which currently delivers highest NMR sensitivity. Other developments will include further increase of sample volumes (currently our volumes are still ≲ 10-fold smaller than in a standard NMR), temperature homogeneity and reduction of MW losses through materials in sample space, which would render the method compatible with low-cost MW sources. On the physical side, the Overhauser mechanism in many heteronuclei is still unexplored. Understanding these mechanistic details will be critical to the reach larger NMR signal enhancements. Nonetheless, we foresee that the simplicity of in-situ DNP will boost future research, in which the full potential of liquid NMR can be exploited.