Elucidation of the folding pathway of a circular permutant of topologically knotted YbeA by tryptophan substitutions

Sarita Puri; Cheng-Yu Liu; I-Chen Hu; Chih-Hsuan Lai; Shang-Te Danny Hsu; Ping-Chiang Lyu

doi:10.1016/j.bbrc.2023.06.021

Elucidation of the folding pathway of a circular permutant of topologically knotted YbeA by tryptophan substitutions

Biochem Biophys Res Commun. 2023 Sep 10:672:81-88. doi: 10.1016/j.bbrc.2023.06.021. Epub 2023 Jun 14.

Authors

Sarita Puri¹, Cheng-Yu Liu², I-Chen Hu², Chih-Hsuan Lai³, Shang-Te Danny Hsu⁴, Ping-Chiang Lyu⁵

Affiliations

¹ Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.
² Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan.
³ Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan; Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan.
⁴ Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan; Institute of Biochemical Sciences, National Taiwan University, Taipei, 10617, Taiwan; International Institute for Sustainability with Knotted Chiral Meta Matter, Hiroshima University, Higashihiroshima, 739-8527, Japan. Electronic address: sthsu@gate.sinica.edu.tw.
⁵ Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, 30013, Taiwan; Department of Medical Science, National Tsing Hua University, Hsinchu, 30013, Taiwan. Electronic address: pclyu@mx.nthu.edu.tw.

PMID: 37343318
DOI: 10.1016/j.bbrc.2023.06.021

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 Å². 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.

Keywords: Circular permutation; Hydrogen-deuterium mass exchange spectrometry; Knotted proteins; Native-like intermediate; Nuclear magnetic resonance spectroscopy; Protein folding.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Circular Dichroism
Escherichia coli*
Kinetics
Protein Folding*
Proteins
Tryptophan

Substances

Proteins
Tryptophan
RlmH protein, E coli