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Do they fold independently of each other or synergistically? If we express these globular domains separately, would their structures remain the same?

I came up with an easy way to test it. Many RNA viruses like HIV and coronaviruses encode multiple proteins in one peptide which folds into a polyprotein with multiple globular domains. The globular domains are then separated by the viral protease. Can we express these proteins separately and compare their structures with native ones? enter image description here enter image description here This question is not a mere curiosity. Many proteins have multiple globular domains which makes structure determination difficult because these domains are very loosely joined together. It would be much easier if we can solve the globular domains individually instead of solving the whole protein at once.

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  • $\begingroup$ I don't think there is any one answer, it probably varies. One data point is the capsid protein of phage p22. If its "I-domain" is removed, the rest of the protein fails to fold. That is maybe more of an intrinsic part of the protein than you meant, though. $\endgroup$
    – timeskull
    Feb 13 at 15:54
  • $\begingroup$ Do they really fold into a polyprotein with a structure that has been determined? I thought that autoproteolysis occurred rapidly. And please provide examples of proteins with multiple domains that make structural determination difficult? In any case, expression and crystallization of parts of proteins is widespread. I certainly wouldn't draw any general conclusions from the polyproteins of RNA viruses. $\endgroup$
    – David
    Feb 13 at 21:24
  • $\begingroup$ Many proteins are shaped like a sausage. For example, the L proteins of nonsegmented -ssRNA viruses have a large RdRp/capping core and a flexible CD-MTase-CTD tail. The L proteins of VSV and rabies are tightly packed which enables us to solve all 4 domains with cryo-EM, while others’ only have the core resolved because the tail is too floppy. annualreviews.org/doi/abs/10.1146/… $\endgroup$
    – 哲煜黄
    Feb 15 at 21:00
  • $\begingroup$ So I came up with an idea. If the CD-MTase-CTD tail can’t be resolved, would it be a better idea to remove it? Cryo-EM solve protein structures by averaging millions of images of identical particles, while the flexible tail only add noise to the images. If the flexible part is removed, the remaining is must easier to align. $\endgroup$
    – 哲煜黄
    Feb 15 at 21:07
  • $\begingroup$ Yes, as David mentioned, it's common to perform x-ray crystallography or Cryo-EM on a truncated version of the protein of interest. NMR is also done on smaller fragments or domains. $\endgroup$
    – timeskull
    Feb 16 at 15:01

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Obviously, this must be considered on a case-by-case basis, so the original poster’s “easy test” is no such thing, relating to viral rather than cellular proteins, and only relevant for the individual cases tested.

However my own impression is that in many, probably most, cases the individual domains fold independently. This can be illustrated by comparing the enzymes of the shikimate pathway in bacteria, such as Escherichia coli, and lower eukaryotes such as Neurospora crassa. The individual proteins are very similar and work in a complex. However in the former case there are separate genes for each enzyme, producing individual polypeptides, whereas in the latter case there is a single gene encoding a multifunctional polypeptide.

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  • $\begingroup$ Another example of a cellular polyprotein is the mammalian Fatty Acid Synthase complex, which I will include in an extended answer that also references the structures determined for some fungal arom complexes. At the moment this answer is a bit thin and doesn't really deserve to have been accepted. $\endgroup$
    – David
    11 hours ago

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