Abstract
The intrinsically disordered ATHP3 was studied at native conditions and in complex with DNA using single amino acid substitutions and high-resolution ion mobility spectrometry coupled to mass spectrometry (trapped IMS-MS). Results showed that ATHP3 can exist in multiple conformations at native conditions (at least 10 conformers were separated), with a variety of proline cis/trans orientations, side chain orientations and protonation sites. When in complex with AT rich DNA hairpins, the -RGRP- core is essential for stabilizing the ATHP3: DNA complex. In particular, the arginine in the sixth position plays an important role during binding to AT-rich regions of hairpin DNA, in good agreement with previous NMR and X-ray data. Mobility based correlation matrices are proposed as a way to reveal differences in structural motifs across the peptide mutants based on the conformational space and relative conformer abundance.
Highlights
Ion mobility spectrometry and mass spectrometry (IMS-MS) have shown advantages for structural biology, and for the study of biomolecules and biomolecular complexes without the influence of the solvent[24,25,26,27]
The peptide sequence influence on the conformational space and DNA binding for the intrinsically disordered ATHP3 is studied using trapped ion mobility spectrometry with collision induced dissociation coupled to mass spectrometry (TIMS-CID-TOF MS) and single amino acid substitutions
These results suggest that R4A/R6A/P7A are directly related to the ATHP3: DNA complex formation
Summary
Ion mobility spectrometry and mass spectrometry (IMS-MS) have shown advantages for structural biology, and for the study of biomolecules and biomolecular complexes without the influence of the solvent[24,25,26,27]. IMS-MS has gained great attention for the study of protein dynamics in their native, partially folded and unfolded forms[24,30,31,32], and when complemented with molecular dynamic simulations, has allowed the identification of specific intramolecular interactions that stabilize conformational states (i.e.; proline and side-chain orientation, protonation site)[33]. The peptide sequence influence on the conformational space and DNA binding for the intrinsically disordered ATHP3 is studied using trapped ion mobility spectrometry with collision induced dissociation coupled to mass spectrometry (TIMS-CID-TOF MS) and single amino acid substitutions. A discussion on the conformational space as a function of the protonation sites, side chain and proline cis/trans orientation and the binding affinity to AT-rich DNA follows
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