15N NMR relaxation studies of backbone dynamics in free and steroid-bound Δ5-3-ketosteroid isomerase from Pseudomonas testosteroni

S. Yun, Soo Jang Do Soo Jang, D. H. Kim, Yong Choi Kwan Yong Choi, Cheon Lee Hee Cheon Lee

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The backbone dynamics of Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been studied in free enzyme and its complex with a steroid ligand, 19-nortestosterone hemisuccinate (19-NTHS), by 15N relaxation measurements. The relaxation data were analyzed using the model-free formalism to extract the model-free parameters (S2,Te, and Rex) and the overall rotational correlation time (Tm). The rotational correlation times were 19.23 ± 0.08 and 17.08 ± 0.07 ns with the diffusion anisotropies (D/D) of 1.26 ± 0.03 and 1.25 ± 0.03 for the free and steroid-bound KSI, respectively. The binding of 19-NTHS to free KSI causes a slight increase in the order parameters (S2) for a number of residues, which are located mainly in helix A1 and strand B4. However, the majority of the residues exhibit reduced order parameters upon ligand binding. In particular, strands B3, B5, and B6, which have most of the residues involved in the dimer interaction, have the reduced order parameters in the steroid-bound KSI, indicating the increased high-frequency (pico- to nanosecond) motions in the intersubunit region of this homodimeric enzyme. Our results differ from those of previous studies on the backbone dynamics of monomeric proteins, in which high-frequency internal motions are typically restricted upon ligand binding.

Original languageEnglish (US)
Pages (from-to)3967-3973
Number of pages7
Issue number13
StatePublished - Apr 3 2001
Externally publishedYes

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