Charge Movement and Structural Changes in the Gas-Phase Unfolding of Multimeric Protein Complexes Captured by Native...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Structural Biology

Background:

• Noncovalent protein complexes can lose native structure in the gas phase due to experimental conditions like energetic collisions.
• The role of charge in gas-phase structural changes of protein complexes is not fully understood.

Purpose of the Study:

• To investigate gas-phase structural changes of alcohol dehydrogenase (ADH) 4mer under varying in-source activation using native top-down mass spectrometry.
• To elucidate the mechanism of charge-directed unfolding in protein complexes.

Main Methods:

• Native top-down (TD) mass spectrometry was employed to analyze alcohol dehydrogenase (ADH) 4mer.
• Electron capture dissociation (ECD) and 193 nm ultraviolet photodissociation (UVPD) were used to induce fragmentation and probe structural changes.
• Analysis of covalent backbone fragments and charge states of UVPD fragments provided insights into unfolding and charge migration.

Main Results:

• ECD fragments indicated unfolding initiated at the N-terminus of ADH.
• UVPD fragment charge states monitored charge migration to unfolded regions.
• A potential 'refolding-after-unfolding' mechanism was observed, supported by hydrogen elimination from radical a-ions, but not in charge-reduced ADH.

Conclusions:

• Results support a charge-directed unfolding mechanism for protein complexes in the gas phase.
• Native TD mass spectrometry provides structure-informative signatures for protein complexes.
• This framework complements other structural characterization techniques like ion mobility and computational modeling.