Our research focuses on two themes.

(i) Dynamic Nuclear Polarization (DNP) and magic-angle spinning (MAS) NMR

The development of MAS NMR for atomic-resolution structural analysis of biological macromolecules appears to be reaching a tipping point. 15 years ago the first de novo structure by MAS NMR of a uniformly labelled tri-peptide was reported. Nowadays it is possible to solve the structures of amyloid fibrils and membrane protein constructs. Unlike X-ray diffraction and solution NMR, MAS NMR does not require macroscopic order and places no limitations on size, complexity, and solubility. With MAS NMR spectroscopy, structural biology has acquired a new tool to address structural questions in previously inaccessible systems.

MAS NMR, however, faces one major challenge: its inherently low sensitivity. Resolving larger and more complex structures requires the acquisition of 2, 3, or even 4-dimensional spectra, but measurement times become, inevitably, too long for these experiments to be feasible. Hence, it is crucial for the continued development of MAS NMR to look for ways to enhance sensitivity.

Dynamic nuclear polarization (DNP) can enhance the sensitivity of NMR by orders of magnitude and has the potential to revolutionize the field. In DNP, paramagnetic species are introduced into the NMR sample. These paramagnetic species (which contain unpaired electrons) can be stable organic radicals, transition metals, or even triplet states and are referred to as "polarizing agents". Microwave irradiation of the NMR sample at or near the electron Larmor frequency transfers the high electron spin polarization to the nuclei and thereby enhances the strength of the NMR signal. Under ideal circumstances, DNP can generate a signal enhancement factor of γe1H = 658 for protons, where γe and γ1H are the gyromagnetic ratios of electrons and protons. 

Polarization, P, is defined as the difference between the populations of the two magnetic sublevels divided by the total number of spins in the system. The MAS NMR spectra are of fibrils formed from the peptide GNNQQNY and were recorded at 400 MHz/263 GHz using 1H-13C cross polarization with and without DNP by Debelouchina et al. PCCP 2010.

MAS NMR studies of complex biological systems require the highest possible resolution and are only feasible at magnetic fields of 16.4 T (700 MHz) and above. Unfortunately, the efficiency of classical DNP decreases with the magnetic field strength. Large methodological advances in the last decade have made DNP/MAS NMR possible up to 21.1 T (900 MHz), but enhancements are still well below the theoretical maximum.

In our group we aim to develop new forms of DNP that can overcome this problem. We follow two approaches.

(a) Photo-DNP

Photo-excitation can generate high, non-Boltzmann polarization in excited triplet states, as well as in stable radicals via chemically induced dynamic electron polarization (CIDEP). We are investigating if and how this polarization can be used for DNP/MAS NMR.  With this approach, using "cold electrons", it is possible to generate enhancement factors considerably larger than 658.

(b) DNP pulse sequences

Both in solution NMR and in MAS NMR, transfer of polarization from 1H to low-gamma nuclei (e.g. 13C and 15N) is a common strategy to increase sensitivity and speed up acquisition. Design principles of pulse sequences that enable this transfer in NMR, are, with certain modifications, also applicable to e--1H polarization transfer in DNP.  We explore both theoretically and experimentally how this is best implemented.


(ii) Advanced EPR spectroscopy of paramagnetic species in biology

(a) Radical chemistry

For a large part of the 20th century, the consensus was among enzymologists that a reaction that could be formulated by two-electron steps, would not proceed via radical intermediates. Today we know that rather the opposite is true. Nature uses and controls the reactivity of unpaired electrons, thereby enabling new reaction pathways. Radical chemistry is abundant in life, even in aerobic environments.

To study radical enzymes, EPR spectroscopy is the method of choice. EPR probes paramagnetic species only, thereby naturally highlighting the exciting chemistry, and provides information on electronic structure. Analysis of EPR spectra, possibly in combination with quantum chemical calculations, allows us to determine the structure of radical intermediates and where they are located. These are vital pieces of information for understanding an enzyme's operating mechanism.

Multi-frequency EPR spectroscopy in combination with isotope labeling enabled the identification of a "thiosulfuranyl" radical intermediate located in the bottom of the active site of E. coli class III ribonucleotide reductase. The observation of this radical provides evidence for the involvement of a thiyl radical in the second, reductive part of the reaction catalyzed by all ribonucleotide reductases. The spectrum (top left) is the frozen solution X-band (9.7 GHz) EPR spectrum of the thiosulfuranyl radical (black), together with a simulation (red). The table shows the different 1H hyperfine interactions for the radical. See also Wei et al. JACS 2014.

(b) High-spin iron sites

Many proteins and enzymes have an iron ion in their active site. EPR spectroscopy is well-suited to probe the electronic structure of iron sites, but its practical use comes with several complications, particularly if the iron is high-spin. First, the EPR experiments are highly demanding and require dedicated instrumentation that is not commercially available. Second, special methods are needed to analyze the spectra. Third, quantum chemical calculations, which enable interpretation of the EPR parameters in terms of the structure of the site, remain highly challenging. Currently we are, in collaboration with Mykhailo Azarkh from the Drescher group, investigating the iron sites in transferrin, a protein that is responsible for iron transport in all vertebrates. We have made crucial steps in acquiring EPR spectra and are now focusing on complete spectral analysis and structural consequences.

Like all members of the transferrin family, human serum transferrin consists of two homologous lobes, termed the N-lobe and the C-lobe. Both lobes bind high-spin Fe3+ avidly. Although the coordinating residues are the same for both lobes, the electronic structure differs for the two Fe3+ sites. This is reflected in their zero-field-splitting parameters, which can only be accurately measured by high-frequency EPR. See also Mathies et al. JBIC2015.