Magnetic Resonance
Magnetic Resonance
Magnetic Resonance
Preprints
https://doi.org/10.5194/mr-2020-31
https://doi.org/10.5194/mr-2020-31

  30 Nov 2020

30 Nov 2020

Review status: a revised version of this preprint is currently under review for the journal MR.

Towards resolving the complex paramagnetic NMR spectrum of small laccase: Assignments of resonances to residue specific nuclei

Rubin Dasgupta, Karthick B. S. S. Gupta, Huub J. M. de Groot, and Marcellus Ubbink Rubin Dasgupta et al.
  • Leiden Institute of Chemistry, University of Leiden, Gorlaeus Laboratory, Einsteinweg 55, 2333 CC, Leiden, The Netherlands

Abstract. Laccases efficiently reduce dioxygen to water in an active site containing a tri-nuclear copper centre (TNC). One reason for its efficiency in catalysis of this complex reaction can be the presence of mobility of active site residues. To probe mobility, NMR spectroscopy is highly suitable. However, several factors complicate the assignment of resonances to active site nuclei in laccases. The paramagnetic nature causes large shifts and line broadening. Furthermore, the presence of slow chemical exchange processes of the imidazole rings of copper ligands result in peak doubling. A third complicating factor is that the enzyme occurs in two states, the native intermediate (NI) and resting oxidized (RO) states, with different paramagnetic properties. The present study aims at resolving the complex paramagnetic NMR spectra of the TNC of Streptomyces coelicolor small laccase (SLAC). With a combination of paramagnetically tailored NMR experiments, all eight His Nδ1 and Hδ1 resonances for the NI state are identified, as well as His Hβ protons for the RO state. With the help of second shell mutagenesis, selective resonances are tentatively assigned to the T2 histidines. This study demonstrates approaches that can be used for sequence specific assignment of the paramagnetic NMR spectra of ligands in the TNC that ultimately may lead to a description of the underlying motions.

Rubin Dasgupta et al.

 
Status: final response (author comments only)
Status: final response (author comments only)
AC: Author comment | RC: Referee comment | SC: Short comment | EC: Editor comment
[Login for authors/editors] [Subscribe to comment alert] Printer-friendly Version - Printer-friendly version Supplement - Supplement

Rubin Dasgupta et al.

Rubin Dasgupta et al.

Viewed

Total article views: 299 (including HTML, PDF, and XML)
HTML PDF XML Total Supplement BibTeX EndNote
220 76 3 299 27 1 1
  • HTML: 220
  • PDF: 76
  • XML: 3
  • Total: 299
  • Supplement: 27
  • BibTeX: 1
  • EndNote: 1
Views and downloads (calculated since 30 Nov 2020)
Cumulative views and downloads (calculated since 30 Nov 2020)

Viewed (geographical distribution)

Total article views: 264 (including HTML, PDF, and XML) Thereof 260 with geography defined and 4 with unknown origin.
Country # Views %
  • 1
1
 
 
 
 
Latest update: 16 Jan 2021
Download