The discovery that some cellular structures behave like droplets and mimic the structure of membraneless organelles is one of the major breakthroughs in modern biology. These structures are formed with the liquid-liquid phase separation and are usually enriched with proteins that contain uncomplexed unstructured regions, which means that the sequence contains low diversity of amino acids.
We have studied the role of the polyalanine region in the human protein PHOX2B and tried to discover how this uncomplexed motif has an impact on the structure and the behavior of the protein. With the help of Alphafold 3.0 and trying different combinations I looked at the structures and interaction between the native protein itself and with its binding DNA. I figured out that the protein mostly is unstructured, with the exception of the α-helical structure of the Homeobox DNA binding domain. A number of pathologic mutations change some energetically favourable interactions to unfavourable, which inhibit the binding of the transcription factor to the promotor sequence but it does not change the structure of the protein itself.
Experimentally we produced the expression of three versions of the protein PHOX2B. Those were the isolated DNA-binding domain, the isolated C-terminal region of the protein and the whole protein with no mutations. We recorded their CD specters before and after thermal denaturation and we also analysed their thermal stability. With that, we confirmed that the native protein PHOX2B and its C-terminal domain are mostly unstructured and without secondary structures. After thermal denaturation we can see that neither the whole protein nor any shorter version of the protein has the same secondary structure as before, but they are now even more unstructured.
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