10 research outputs found
Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances
Deuterium-Hydrogen Exchange Coupled With Mass Spectrometry Revealed A Novel Autoantibody Binding Epitope and Substrate Recognition Site In ADAMTS13 Protease,
Helical structure and stability in human apolipoprotein A-I by hydrogen exchange and mass spectrometry
Apolipoprotein A-I (apoA-I) stabilizes anti-atherogenic high density lipoprotein particles (HDL) in the circulation and governs their biogenesis, metabolism, and functional interactions. To decipher these important structureâfunction relationships, it will be necessary to understand the structure, stability, and plasticity of the apoA-I molecule. Biophysical studies show that lipid-free apoA-I contains a large amount of α-helical structure but the location of this structure and its properties are not established. We used hydrogen-deuterium exchange coupled with a fragmentation-separation method and mass spectrometric analysis to study human lipid-free apoA-I in its physiologically pertinent monomeric form. The acquisition of â100 overlapping peptide fragments that redundantly cover the 243-residue apoA-I polypeptide made it possible to define the positions and stabilities of helical segments and to draw inferences about their interactions and dynamic properties. Residues 7â44, 54â65, 70â78, 81â115, and 147â178 form α-helices, accounting for a helical content of 48 ± 3%, in agreement with circular dichroism measurements (49%). At 3 to 5 kcal/mol in free energy of stabilization, the helices are far more stable than could be achieved in isolation, indicating mutually stabilizing helix bundle interactions. However the helical structure is dynamic, unfolding and refolding in seconds, allowing facile apoA-I reorganization during HDL particle formation and remodeling
Protein FoldingâHow and Why: By Hydrogen Exchange, Fragment Separation, and Mass Spectrometry
Effects of the Iowa and Milano Mutations on Apolipoprotein AâI Structure and Dynamics Determined by Hydrogen Exchange and Mass Spectrometry
The Iowa point mutation in apolipoprotein A-I (G26R)
leads to a
systemic amyloidosis condition, and the Milano mutation (R173C) is
associated with hypoalphalipoproteinemia, a reduced plasma level of
high-density lipoprotein. To probe the structural effects that lead
to these outcomes, we used amide hydrogenâdeuterium exchange
coupled with a fragment separation/mass spectrometry analysis (HX
MS). The Iowa mutation inserts an arginine residue into the nonpolar
face of an α-helix that spans residues 7â44 and causes
changes in structure and structural dynamics. This helix unfolds,
and other helices in the N-terminal helix bundle domain are destabilized.
The segment encompassing residues 116â158, largely unstructured
in wild-type apolipoprotein A-I, becomes helical. The helix spanning
residues 81â115 is destabilized by 2 kcal/mol, increasing the
small fraction of time it is transiently unfolded to â„1%, which
allows proteolysis at residue 83 in vivo over time, releasing an amyloid-forming
peptide. The Milano mutation situated on the polar face of the helix
spanning residues 147â178 destabilizes the helix bundle domain
only moderately, but enough to allow cysteine-mediated dimerization
that leads to the altered functionality of this variant. These results
show how the HX MS approach can provide a powerful means of monitoring,
in a nonperturbing way and at close to amino acid resolution, the
structural, dynamic, and energetic consequences of biologically interesting
point mutations