Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Structural Biology

Background:

• Protein conformation is crucial for biological function.
• Understanding protein dynamics in solution is challenging.
• Existing methods may have limitations in probing subtle conformational shifts.

Purpose of the Study:

• To introduce and validate hydrogen-exchange electrospray-ionization mass spectrometry (H-dX-ESI-MS) as a method for studying protein conformational changes.
• To demonstrate the applicability of H-dX-ESI-MS in analyzing protein structural dynamics in response to environmental stimuli.
• To investigate methanol-induced conformational changes in bovine ubiquitin.

Main Methods:

• Utilizing electrospray-ionization mass spectrometry (ESI-MS) to measure hydrogen/deuterium exchange.
• Implementing H-dX-ESI-MS to quantify deuterium incorporation into proteins over time.
• Analyzing changes in deuterium uptake patterns to infer conformational alterations.
• Applying the method to bovine ubiquitin in aqueous acidic solutions with varying methanol concentrations.

Main Results:

• H-dX-ESI-MS effectively detected and quantified hydrogen/deuterium exchange in bovine ubiquitin.
• The method revealed distinct deuterium uptake profiles corresponding to different protein conformers.
• Methanol addition induced measurable changes in the hydrogen-deuterium exchange patterns of ubiquitin, indicating conformational shifts.
• The results demonstrate the sensitivity of H-dX-ESI-MS to subtle structural rearrangements.

Conclusions:

• Hydrogen-exchange electrospray-ionization mass spectrometry is a powerful and effective technique for probing protein conformational changes in solution.
• The method provides valuable insights into protein dynamics and structural responses to environmental factors.
• H-dX-ESI-MS holds significant potential for future studies in structural biology and drug discovery.