Using mass spectrometry to study copper-protein binding under native and non-native conditions: beta-2-microglobulin

Area of Science:

• Biochemistry
• Proteomics
• Analytical Chemistry

Background:

• Beta-2-microglobulin (beta2m) destabilization and amyloid fiber formation are linked to copper(II) (Cu(II)) presence.
• High affinity of Cu(II) for unfolded beta2m states is suspected to drive protein destabilization.
• Traditional methods like NMR and X-ray crystallography face challenges in determining Cu(II) binding sites in native and unfolded beta2m due to aggregation and denaturant use.

Purpose of the Study:

• To determine the specific Cu(II) binding sites in both native and unfolded conformations of beta-2-microglobulin (beta2m).
• To overcome limitations of traditional techniques for studying Cu(II)-protein interactions in challenging protein states.

Main Methods:

• Metal-catalyzed oxidation (MCO) reactions coupled with mass spectrometry (MS) were employed.
• The MCO/MS approach allowed analysis at low protein concentrations to prevent amyloid formation.
• The method was effective even in the presence of high denaturant concentrations (8 M urea).

Main Results:

• Cu(II) coordination in native beta2m involves the N-terminus and His31.
• In the unfolded state (8 M urea), Cu(II) binds to the N-terminus, His31, His51, and His81.
• These findings reveal distinct Cu(II) coordination structures in native versus unfolded beta2m.

Conclusions:

• The MCO/MS method successfully identified Cu(II) binding sites in beta2m, overcoming limitations of traditional techniques.
• The study confirms a well-defined, yet different, Cu(II) coordination structure in unfolded beta2m, explaining its increased affinity.
• MCO/MS is a valuable tool for characterizing metal-binding sites in proteins that are difficult to study otherwise.