Probing Structural and Motional Features of the C-Terminal Part of the Human Centrin 2/P17-XPC Microcrystalline Complex by Solid-State NMR Spectroscopy

Abstract

Insight into structural and motional features of the Cterminal part of the Human Centrin 2 in complex with the peptide P17-XPC was obtained by using complementary solid-state NMR methods. We demonstrate that the experimental conditions and procedures of sample crystallization determine the quality of solid-state NMR spectra and the internal mobility of the protein. 13 C− 13 C and 15 N− 15 N correlation spectra reveal intra-and inter-residue dipolar connectivities and provide partial, site-specific assignments of 13 C and 15 N resonance signals. The secondary structure of the C-ter HsCen2/P17-XPC complex in a microcrystalline state appears similar to that found in solution. Conformational flexibility is probed through relaxation-compensated measurements of dipolar order parameters that exploit the dynamics of cross-polarization in multidimensional experiments. The extracted dipolar coupling constants and relevant order parameters reveal increased backbone flexibility of the loops except for residues involved in coordination with the Ca 2+ cation that stabilizes the hydrophobic pocket containing the peptide P17-XPC.

Quantitative one- and two-dimensional 13C spectra of microcrystalline proteins with enhanced intensity

“…We have demonstrated that recoupling schemes such as Phase Alternated Recoupling Irradiation Schemes (PARIS) (Weingarth et al 2009b, c) and PARIS-xy (Weingarth et al 2010) permit one to reach similar peak amplitudes (±10 %) for chemically different sites on time scales as short as a few hundreds of milliseconds. We have previously used these pulse schemes to record sensitive 2D correlation spectra of microcrystalline proteins (Weingarth et al 2009c;Herbert-Pucheta et al 2012b), amyloid fibrils (Weingarth et al 2011b) and mixtures of crystallographic forms (Herbert-Pucheta et al 2011), and to restore the symmetry in 2D homonuclear correlation experiments of simple amino acids (Herbert-Pucheta et al 2012a).…”