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Immobilized proteins in porous materials – Structural studies by Pulse EPR dipolar spectroscopY

Periodic Reporting for period 1 - iSPY (Immobilized proteins in porous materials – Structural studies by Pulse EPR dipolar spectroscopY)

Reporting period: 2018-06-01 to 2020-05-31

Nature is by far the most versatile chemist and modern research efforts have harnessed the power of Nature by using biomolecules such as proteins as building blocks or targets for various technological applications which have important societal impact such as medical diagnostics, pollution detection and catalysis. In many cases the immobilization of a protein in a synthetic matrix is essential. In particular protein-porous material hybrids have received much attention but their preparation have been non-trivial, often limited by the size compatibility between the pore and the protein as well as the surface properties. The quest for a suitable protein-matrix combination not only requires extensive synthetic optimization, but also the development of appropriate methodologies that can be used to determine the effect of the matrix on the structure and stability of the protein. In this multidisciplinary action, the stabilities, structures and dynamics of heme proteins (globins) immobilized in mesoporous silica or titania materials were studied by electron paramagnetic resonance (EPR, also known as electron spin resonance, ESR). This class of hybrid materials are themselves also of great interest because of potential electrochemical biosensing and biocatalysis applications. Spin-labeled globin proteins were prepared and incorporated into (modified) mesoporous silica and titania materials. Advanced pulse EPR methods were used to measure distances on the nanometer scale within the free and immobilized globin proteins. Combined with computational models, these measurements provided unique insights into effects of incorporation on the tertiary structures and conformational flexibilities of the proteins. This action not only result in the development of a generic analytical toolbox, based on spin-label EPR, for the characterization of proteins immobilized in matrices, but also lead to advances in the understanding and preparation of protein-porous material hybrids as well as other paramagnetic materials.
i.) Hybrid materials consisting of spin-labeled globin proteins incorporated into (modified) mesoporous silica and titania materials have been prepared and studied by EPR techniques. The results offer new insights into the incorporation process and a set of methodologies for achieving this. These have been disseminated via two workshops, an international conference, and manuscripts are in preparation.
ii.) Surface-modified titania materials have been prepared and studied by EPR and spin-probe EPR. The results offer new insights into the surface modification process which will guide the development of more advanced materials. These have been disseminated via a publication, others are in preparation and also on Twitter.
iii.) Other paramagnetic materials, including bitumen and porous metal-organic frameworks for gas storage, have also been studied by EPR. The results offer new insights into the composition and the degradation mechanism of such materials with important industrial implications. These have been disseminated via a publication, others are in preparation and also on Twitter. The bituminous research involved collaborators from the private sector who are interested in further exploration.
iv.) In situ spectroelectrochemical (EPR coupled with electrochemistry) setups have been built and tested. The result is the development of methodologies, which not only have the potential to be used for studying hybrid materials under working conditions, but also electrochemical processes in general. These have been disseminated via a publication, other manuscripts are in preparation and also on Twitter.
This project has expanded our knowledge of:
i.) How proteins behave within hybrid materials. These results will impact the preparation of new hybrid materials with improved properties, expanding their potential utility in technological applications such as biosensors, biofuel cells and biocatalysts.
ii.) Surface-modified titania materials. Aside from their valorization during the preparation of hybrid materials, these results will also impact the preparation of titania materials in general, which will aid the development of better functional materials such membranes for separation technologies and photocatalyst.
iii.) The composition and degradation of paramagnetic materials with existing and potential industrial applications. These results will potentially impact on the production and maintenance of such materials.
Ultimately these results will improve the lives of people and address some of the ecological and economical demands of the 21st century.

Furthermore, this project has also advanced the state of the art with regards to EPR methods for the characterization of paramagnetic materials in general. These methods offer unique information regarding the materials used in numerous fields of studies such as those listed in i.), ii.) and iii.). This project will potentially fuel the further development of EPR methods.
overview of project