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

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.