Solid-state NMR approaches to deliver new insights into the extracellular matrix

The extracellular matrix (ECM) contains important chemical and mechanical cues to shape and alter cell behaviour. However, there are limited tools for analysing the structures and structural interactions present at the atomic length scale within the intact ECM.

Solid-state nuclear magnetic resonance (ssNMR) can probe atomic-level structures within intact ECM samples without extensive purification and extraction. We have developed an approach for investigating collagen proteins within the ECM using ssNMR [1] and are interested in extending our approach to other ECM glycoproteins, such as laminin or fibronectin. Once extended to other ECM components, we expect to be able to correlate the atomic-level structures to the mechanical properties of the ECM [2]. Such insight can provide new design principles for ECM-mimicking materials, such as biomaterials for regenerative medicine.

In previous work [3], we proposed a new pathway for collagen/cartilage ECM degradation via radical species that are centred on glycine residues in proteins, in contrast to the ‘free’ radicals that are commonly implicated in mechanisms of ageing. We are interested in developing ssNMR and potentially electron paramagnetic resonance (EPR) approaches for investigating this new source of oxidative stress in collagen-containing ECMs and tissues.

We also have an interest in interactions of the microbial cell walls (fungal or bacterial) with mammalian ECM components. SsNMR provides a new approach for probing such interactions that can provide structural information complementary to other techniques, such as microscopy, biochemical assays, and modelling.

We are looking for students with a strong biological/biochemical background who are interested in undertaking a unique and interdisciplinary project, gaining hands-on experience in using physical science techniques to address biological problems.

Objectives

• Develop an approach for investigating specific ECM proteins or glycoproteins using ssNMR
• Applying the ssNMR approach to deliver new insights on cell culture-derived ECM and eventually tissue biopsies
• Using ssNMR and EPR to probe glycine radicals and understand conditions of their formation within the ECM
• Using ssNMR to probe microbial cell walls and their interaction with mammalian ECM components

Techniques and methodologies that the student will learn:

• ECM production via cell culture
• Stable isotope enrichment
• Solid-state nuclear magnetic resonance (NMR) spectroscopy, including data acquisition and analysis

Additional techniques and methodologies, as required by the project:

• Electron microscopy
• Mass-spectrometry based proteomics
• Electron paramagnetic resonance (EPR) spectroscopy
• Atomic force microscopy (AFM)
• Protein expression and purification
• Bioinformatics
• 
  

References:

1. Chow, W. Ying, Rakesh Rajan, Karin H. Muller, David G. Reid, Jeremy N. Skepper, Wai Ching Wong, Roger A. Brooks, et al. 2014. NMR spectroscopy of native and in vitro tissues implicates polyADP ribose in biomineralization. Science 344: 742–746. https://doi.org/10.1126/science.1248167.
2. Chow, Wing Ying, Chris J. Forman, Dominique Bihan, Anna M. Puszkarska, Rakesh Rajan, David G. Reid, David A. Slatter, et al. 2018. Proline provides site-specific flexibility for in vivo collagen. Scientific reports 8: 13809. https://doi.org/10.1038/s41598-018-31937-x.
3. Chow, Wing Ying, Brendan P. Norman, Norman B. Roberts, Lakshminarayan R. Ranganath, Christian Teutloff, Robert Bittl, Melinda J. Duer, James A. Gallagher, and Hartmut Oschkinat. 2020. Pigmentation Chemistry and Radical-Based Collagen Degradation in Alkaptonuria and Osteoarthritic Cartilage. Angewandte Chemie 59: 11937–11942. https://doi.org/10.1002/anie.202000618.

BBSRC Strategic Research Priority: Understanding the rules of life – Structural Biology, and Microbiology, and Integrated Understanding of Health - Pharmaceuticals, Regenerative Biology, and Ageing.

Techniques that will be undertaken during the project:

• ECM production via cell culture
• Stable isotope enrichment
• Solid-state nuclear magnetic resonance (NMR) spectroscopy, including data acquisition and analysis

Additional techniques and methodologies, as required by the project:

• Electron microscopy
• Mass-spectrometry based proteomics
• Electron paramagnetic resonance (EPR) spectroscopy
• Atomic force microscopy (AFM)
• Protein expression and purification
• Bioinformatics