Abstract
Glial fibrillary acidic protein (GFAP) is a promising biomarker for brain and spinal cord disorders. Recent studies have highlighted the differences in the reliability of GFAP measurements in different biological matrices. The reason for these discrepancies is poorly understood as our knowledge of the protein's 3-dimensional conformation, proteoforms, and aggregation remains limited. Here, we investigate the structural properties of GFAP under different conditions. For this, we characterized recombinant GFAP proteins from various suppliers and applied hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a snapshot of the conformational dynamics of GFAP in artificial cerebrospinal fluid (aCSF) compared to the phosphate buffer. Our findings indicate that recombinant GFAP exists in various conformational species. Furthermore, we show that GFAP dimers remained intact under denaturing conditions. HDX-MS experiments show an overall decrease in H-bonding and an increase in solvent accessibility of GFAP in aCSF compared to the phosphate buffer, with clear indications of mixed EX2 and EX1 kinetics. To understand possible structural interface regions and the evolutionary conservation profiles, we combined HDX-MS results with the predicted GFAP-dimer structure by AlphaFold-Multimer. We found that deprotected regions with high structural flexibility in aCSF overlap with predicted conserved dimeric 1B and 2B domain interfaces. Structural property predictions combined with the HDX data show an overall deprotection and signatures of aggregation in aCSF. We anticipate that the outcomes of this research will contribute to a deeper understanding of the structural flexibility of GFAP and ultimately shed light on its behavior in different biological matrices.
| Original language | English |
|---|---|
| Pages (from-to) | 649-664 |
| Number of pages | 16 |
| Journal | Proteins |
| Volume | 92 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - May 2024 |
Bibliographical note
© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.Funding
Research of DG, CT, and SA are supported by the European Commission (Marie Curie International Training Network, grant agreement No 860197 (MIRIADE)). CT is supported by JPND (bPRIDE), Health Holland, the Dutch Research Council (ZonMW), Alzheimer Drug Discovery Foundation, The Selfridges Group Foundation, Alzheimer Netherlands, and Alzheimer Association. CT is the recipient of ABOARD, which is a public\u2010private partnership receiving funding from ZonMW (#73305095007) and HealthHolland, Topsector Life Sciences & Health (PPP\u2010allowance; #LSHM20106). More than 30 partners participate in ABOARD. ABOARD also receives funding from Edwin Bouw Fonds and Gieskes\u2010Strijbisfonds. CT has a collaboration contract with ADx Neurosciences, Quanterix and Eli Lilly, performed contract research or received grants from AC\u2010Immune, Axon Neurosciences, Biogen, Brainstorm Therapeutics, Celgene, EIP Pharma, Eisai, Olink, PeopleBio, Roche, Toyama, Vivoryon. CT serves on editorial boards of Medidact Neurologie/Springer, Alzheimer Research and Therapy, Neurology: Neuroimmunology & Neuroinflammation, and is editor of a Neuromethods book Springer. The rest of the authors do not have any competing interests to declare.
| Funders | Funder number |
|---|---|
| Alzheimer's Association | |
| Health Holland | |
| Edwin Bouw Fonds and Gieskes‐Strijbisfonds | |
| Nederlandse Organisatie voor Wetenschappelijk Onderzoek | |
| EU Joint Programme – Neurodegenerative Disease Research | |
| Eli Lilly and Company | |
| Topsector Life Sciences & Health | |
| ZonMw | |
| HealthHolland | |
| Alzheimer's Drug Discovery Foundation | |
| European Commission | 860197 |
Keywords
- Humans
- Deuterium Exchange Measurement/methods
- Glial Fibrillary Acidic Protein/chemistry
- Phosphates
- Protein Conformation
- Reproducibility of Results
- Recombinant Proteins