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Investigation of D76N β2-Microglobulin Using Protein Footprinting and Structural Mass Spectrometry.

Owen Cornwell1, James R Ault2, Nicholas J Bond1

  • 1Biopharmaceuticals R & D, AstraZeneca, Granta Park, Cambridge CB21 6GP, U.K.

Journal of the American Society for Mass Spectrometry
|February 15, 2021
PubMed
Summary

The D76N variant of beta2-microglobulin (β2m) shows increased aggregation despite similar structures. Complementary protein footprinting methods revealed subtle differences in conformational dynamics, particularly in the E-F loop, explaining the aggregation propensity.

Keywords:
D76NFPOPHDXamyloidprotein conformationstructural mass spectrometryβ2m

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Dynamics

Background:

  • Beta2-microglobulin (β2m) is a 99-residue amyloidogenic protein implicated in various diseases.
  • A specific variant, D76N β2m, exhibits significantly higher aggregation propensity compared to the wild-type protein.
  • Previous structural studies using NMR and X-ray crystallography showed no discernible structural differences between wild-type and D76N β2m, leaving the cause of aggregation disparity unexplained.

Purpose of the Study:

  • To investigate the subtle differences in conformational dynamics between wild-type β2m and the aggregation-prone D76N variant.
  • To elucidate the molecular basis for the increased aggregation propensity of the D76N β2m variant.
  • To demonstrate the utility of combining multiple protein footprinting techniques for analyzing highly similar proteins.

Main Methods:

  • Employed two complementary protein footprinting techniques: hydrogen-deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins mass spectrometry (FPOP-MS).
  • Utilized ion mobility-mass spectrometry to enhance the analysis of conformational dynamics.
  • Focused on probing differences in the E-F loop region, where the D76N substitution occurs.

Main Results:

  • HDX-MS revealed significantly higher deuterium uptake in the E-F loop of the D76N variant, indicating increased solvent accessibility and/or reduced hydrogen bonding.
  • FPOP-MS showed only minimal differences in oxidation levels within the E-F loop, suggesting limited large-scale side-chain movements.
  • The combined data suggest a perturbation of the hydrogen-bonding network within the E-F loop of the D76N variant.

Conclusions:

  • The D76N substitution in β2m leads to subtle but significant alterations in the protein's conformational dynamics, specifically affecting the hydrogen-bonding network in the E-F loop.
  • These dynamic differences, rather than static structural changes, likely account for the increased aggregation propensity of the D76N variant.
  • The study highlights the power of integrating complementary biophysical techniques like HDX-MS and FPOP-MS to uncover functionally relevant, subtle differences between closely related protein structures.