The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as ‘AT-hooks’, are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three ‘AT-hook’ peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, ‘key-locked’ conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution.
Note: When clicking on a Digital Object Identifier (DOI) number, you will be taken to an external site maintained by the publisher.
Some full text articles may not yet be available without a charge during the embargo (administrative interval).
What is a DOI Number?
Some links on this page may take you to non-federal websites. Their policies may differ from this site.
-
more » « less
Abstract -
more » « less
Rationale The molecular environment is known to impact the secondary and tertiary structures of biomolecules both in solution and in the gas phase, shifting the equilibrium between different conformational and oligomerization states. However, there is a lack of studies monitoring the impacts of solution additives and gas‐phase modifiers on biomolecules characterized using ion mobility techniques.
Methods The effect of solution additives and gas‐phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100–500 ms) using nanoelectrospray ionization coupled to trapped ion mobility spectrometry with detection by time‐of‐flight mass spectrometry. Organic compounds used as additives/modifiers (methanol, acetonitrile, acetone) were either added to the aqueous protein solution before ionization or added to the ion mobility bath gas by nebulization.
Results Changes in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) and the protein. In the presence of gas‐phase modifiers (i.e., N2doped with methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas‐modifier mass and size and changes in the relative abundances and number of IMS bands are observed.
Conclusions We attribute the observed changes in the mobility profiles in the presence of gas‐phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.
-
more » « less
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. -
