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\begin{document}

\title{Structural flexibility and heterogeneity of recombinant human glial
fibrillary acidic protein (GFAP)}



\author[1]{Dea Gogishvili}%
\author[2]{Eva Illes-Toth}%
\author[2]{Matthew Harris J}%
\author[2]{Christopher Hopley}%
\author[3]{Charlotte Teunissen E}%
\author[1]{Sanne Abeln}%
\affil[1]{Vrije Universiteit Amsterdam Afdeling Informatica}%
\affil[2]{LGC Limited}%
\affil[3]{Amsterdam Neuroscience}%


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  \date{May 17, 2024}


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\begin{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
characterised 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
CSF 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 artificial CSF overlap with
predicted conserved dimeric interface regions. Our results suggest that
GFAP exists in several conformational forms. 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
behaviour in different biological matrices.%
\end{abstract}%



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\textbf{Hosted file}

\verb`HDX_RawData.xlsx`  available at \url{https://authorea.com/users/671285/articles/670903-structural-flexibility-and-heterogeneity-of-recombinant-human-glial-fibrillary-acidic-protein-gfap}

\textbf{Hosted file}

\verb`HDX_SummaryTable.xlsx`  available at \url{https://authorea.com/users/671285/articles/670903-structural-flexibility-and-heterogeneity-of-recombinant-human-glial-fibrillary-acidic-protein-gfap}

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