Active Resonator Development for nano-EPR of single crystal proteins

Informacje na temat projektu

Act-EPR

Identyfikator umowy o grant: 745702

DOI

10.3030/745702

Projekt został zamknięty

Data podpisania przez KE 16 Marca 2017

Data rozpoczęcia 1 Maja 2017

Data zakończenia 23 Czerwca 2019

Finansowanie w ramach

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Koszt całkowity

€ 171 460,80

Wkład UE

€ 171 460,80

Koordynowany przez

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Germany

Periodic Reporting for period 1 - Act-EPR (Active Resonator Development for nano-EPR of single crystal proteins)

Okres sprawozdawczy: 2017-05-01 do 2019-04-30

Podsumowanie kontekstu i ogólnych celów projektu

In order to keep up with societal challenges of the 21st century, we must devise sustainable ways to efficiently store and retrieve energy. What better way to solve these problems than to look to nature? Fully understanding the catalytic mechanism of enzymes provides a basis for synthetic models designed for practical applications. For example, hydrogenase based and inspired systems provide an interesting route to advance towards a “hydrogen economy” and the future of clean energy.

We employ Electron Paramagnetic Resonance (EPR) to study the paramagnetic states of hydrogenases and obtain information on electronic and geometrical structure of their active site. Single crystal experiments are the ultimate method in determining the full-tensor magnetic interactions of the enzyme. However, the application of single-crystal EPR to metallo-proteins is severely limited by the small crystals sizes which are less than 27 nL in volume. In order to make single crystal EPR available as tool to study redox and metallo-proteins, a breakthrough in absolute sensitivity is necessary. The development of a self-resonant micro-helix (Fig02) with a factor up to 28 in signal-to-noise improvement has met this goal. It is now possible to perform advanced pulse EPR experiments on protein single-crystals with dimensions that are typical for X-ray crystallography diffraction. This has a direct impact on biophysical and biochemical basic science initiatives.

For the first time, a full g-tensor is proposed for the Hox state of [FeFe]-hydrogenase (Fig01). The protein single-crystal had dimensions of 0.3 x 0.1 x 0.1 mm3 (3 nL) and each trace took only 8 minutes to collect with a signal-to-noise ratio of 290. The g-tensor proposed here is directly measured and refines the previously proposed g-tensors which relied heavily on assumptions. Act-EPR is at the core of basic science research in order to better understand both the structure and function of enzymes. Not only does the understanding of such enzymes advance our scientific knowledge, but it provides a path for drug discovery and bioengineering mimics of enzymes for industrial purposes.

Prace wykonane od początku projektu do końca okresu sprawozdawczego oraz najważniejsze dotychczasowe rezultaty

Within Work Package 1 the Milestone M1 and Deliverable D1 were completed. The micro-helix geometry and coupling mechanism (Fig 02) meet the goal of Milestone M4 and meet the first half of Deliverable D2 since it can be reproduced quickly and fabricated with a frequency within the range of the commercial X-band bridges (9.3–9.8 GHz). Using the developed fabrication jig, reproducibility within this range is guaranteed. The micro-helix is coupled by a PC board strip-line inductive coupler on Teflon substrate which is manufactured by standard lithography techniques. Comparing the commercially available resonators (Bruker dielectric MD5W1 and split-ring MS3) and state-of-the-art planar micro-resonators (PMR) to the self-resonant micro-helix using a standard test sample. For samples that saturate with microwave power, such as proteins, a factor of 6 is achieved compared to commercially available resonators. However, for samples that do not saturate with magnetic power, such as those used in quantum computing, a factor of 28 is achieved compared to commercially available resonators. Since this fellowship is only concerned with samples that saturate with the microwave power, this signal-to-noise improvement constitutes a factor of 36 reduction in measurement time.

From this technical advancement we have completed the Milestone M5 and M6 by being the first to measure an EPR signal on [FeFe]-hydrogenase single crystal. These experiment allowed for the measurement of hyperfine- and quadrupole-information which gives insight into the catalytic mechanisms of the enzyme.

Exploitation of the results is at its early stages. These results have been accepted in the high impact journal Science Advances. The PR team at the Max Planck Institute for Chemical Energy Conversion are preparing press statements. We are working together to create a video explaining these results and their importance to the biophysical community. Although these results are of a basic science nature, it is important to also explain the importance of this research to the public. This remains a challenge and we are actively searching for innovative ways to disseminate basic science research.

For further details see the final report.

Innowacyjność oraz oczekiwany potencjalny wpływ (w tym dotychczasowe znaczenie społeczno-gospodarcze i szersze implikacje społeczne projektu)

The completion of the self-resonant micro-helix (Fig. 02) opens up the possibility to study single-crystals with paramagnetic intermediates on crystals of dimensions less than 0.3 x 0.3 x 0.3 mm^3. These dimensions allow for studying protein crystals at the same volumes needed for structure determining x-ray crystallography diffraction experiments. The combination of structure and function determination is highly informative and highly sought after. Due to the very high efficiency parameter, the micro-helix geometry is advantageous in extending pulse EPR to experiments that usually require costly high-powered microwave amplifiers, further expanding the applicability of pulse EPR.

Although we have applied the micro-helix to study hydrogenase, the instrument is not limited to proteins. In fact, there are many fields of research, such as, molecular magnets, quantum computing, and material science which can benefit from the micro-helix geometry and EPR in general. To further the usefulness of the micro-helix for quantum computing, a micro-helix has been constructed of super conducting NbTi wire. The resonator is in the early stages of development but the increase in magnetic field will allow new quantum computing experiments.

This project maintains the long-term goal of studying enzymes structure and function which could not be previously studied due to lack of sensitivity. The self-resonant micro-helix has enabled, for the first time, the collection of EPR data from a 0.3 x 0.1 x 0.1 mm3 (3 nL) single crystal of [FeFe]-hydrogenase from Clostridium pasteurianum (CpI; [6Fe]-cluster) in the Hox state and the determination of the g-tensor (Fig01). Additionally, advanced pulse methods that measure the hyperfine coupling could be collected from the same protein single-crystal. The determination of the g-tensor and the ability to perform hyperfine experiments has an impact in both analytical and bioinorganic chemistry. Fundamentally understanding such enzymes is of broad biochemical and biophysical importance as we move towards bioengineering mimics of nature’s most elusive chemistry.

Currently crystals of suitable size (between 3-5 nL) are available of the [FeFe]-hydrogenase CpI in the Hox state and a reduced CpI-apo which has an EPR signal derived from the reduced four iron-sulfur clusters. In CpI-apo, the active center (H-cluster) is not present. The CPI-apo crystal could be used to study the electron transfer pathway of the [FeFe]-hydrogenase and how it relates to the function of the hydrogenase. It is also possible to obtain crystals in the inactive Hox-CO state, which may lend insight into reducing the oxygen sensitivity of the [FeFe]-hydrogenase. These samples will be studied in detail.

It is wholly expected to continue this line of research to maximize the socio-economic impact of this basic science research. Specifically in order to study the key roadblocks to creating the hydrogen economy using hydrogenase or similar enzymes. Some roadblocks include oxygen tolerance, miniaturisation of the protein backbone, and overall turn over rate.

Figure 1. Data collected for FeFe-Hydrogenase

Figure 2. microHelix geometry

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