AI Article Synopsis
- CP74 is a modified version of a protein from E. coli that untangles a complex knotted structure and forms a stable dimer through domain swapping, revealing a large interaction area between the two protein units.* -
- To study how changes in tryptophan residues affect the protein's structure and stability, researchers replaced five tryptophans with phenylalanine and used various biophysical methods to assess the impact.* -
- Findings showed that overall structure remained stable despite single substitutions, while detailed analysis indicated that specific tryptophan residues, especially W100, are crucial for CP74's proper folding and stability, highlighting the significance of the hinge region in maintaining its structure.*
Article Abstract
CP74 is an engineered circular permutant of a deep trefoil knotted SpoU-TrmD (SPOUT) RNA methyl transferase protein YbeA from E. coli. We have previously established that the circular permutation unties the knotted topology of YbeA and CP74 forms a domain-swapped dimer with a large dimeric interface of ca. 4600 Å. To understand the impact of domain-swapping and the newly formed hinge region joining the two folded domains on the folding and stability of CP74, the five equally spaced tryptophan residues were individually substituted into phenylalanine to monitor their conformational and stability changes by a battery of biophysical tools. Far-UV circular dichroism, intrinsic fluorescence, and small-angle X-ray scattering dictated minimal global conformational perturbations to the native structures in the tryptophan variants. The structures of the tryptophan variants also showed the conservation of the domain-swapped ternary structure with the exception that the W72F exhibited significant asymmetry in the α-helix 5. Comparative global thermal and chemical stability analyses indicated the pivotal role of W100 in the folding of CP74 followed by W19 and W72. Solution-state NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry further revealed the accumulation of a native-like intermediate state in which the hinge region made important contributions to maintain the domain-swapped ternary structure of CP74.
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| http://dx.doi.org/10.1016/j.bbrc.2023.06.021 | DOI Listing |
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