Site-specific incorporation of 19F-nulcei at protein C-terminus to probe allosteric conformational transitions of metalloproteins
Allosteric conformational change is an important paradigm in the regulation of protein function, which is typically triggered by the binding of small cofactors, metal ions or protein partners. Here, we found those conformational transitions can be effectively monitored by 19 F NMR, facilitated by a...
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Veröffentlicht in: | Communications biology 2024-12, Vol.7 (1), p.1613-12 |
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Sprache: | eng |
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Zusammenfassung: | Allosteric conformational change is an important paradigm in the regulation of protein function, which is typically triggered by the binding of small cofactors, metal ions or protein partners. Here, we found those conformational transitions can be effectively monitored by
19
F NMR, facilitated by a site-specific
19
F incorporation strategy at the protein C-terminus using asparaginyl endopeptidase (AEP). Three case studies show that C-terminal
19
F-nuclei can reveal protein dynamics not only adjacent but also distal to C-terminus, including those occurring in a hemoprotein neuroglobin (Ngb), calmodulin (CaM), and a cobalt metalloregulator (CoaR) responding to both cobalt and tetrapyrrole. In Ngb, the heme orientation disorder is affected by missense mutations that perturb backbone rigidity or surface charges close to the heme axial ligands. In CaM, the C-terminal
19
F-nuclei is an ideal probe for detecting the binding states of Ca
2+
, peptides and inhibitors. Furthermore, multiple
19
F-moieties were incorporated into the two domains of CoaR, revealing the intrinsically disordered C-terminal metal binding tail might be an allosteric conformational switch to maintain cobalt homeostasis and balance corrinoid biosynthesis. This study demonstrates that the AEP-based
19
F-modification strategy can be applied to various targets to study allosteric regulation, especially for those biological processes modulated by the protein C-terminus.
The site-specific
19
F labeling at protein C-terminus catalyzed by
Oa
AEP1
C247A
is an efficient approach to utilize
19
F NMR, which is compatible with the genetic code expansion technology. Three cases studies show this approach is suitable for probing allosteric conformational transitions in metalloproteins. |
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ISSN: | 2399-3642 2399-3642 |
DOI: | 10.1038/s42003-024-07331-x |