Protein conformational changes determined by matrix-assisted laser desorption mass spectrometry

Area of Science:

• Biochemistry
• Protein Chemistry
• Analytical Chemistry

Background:

• Protein denaturation involves complex conformational changes.
• Understanding these changes is crucial for various biological processes.
• Existing methods may not fully capture sequential unfolding events.

Purpose of the Study:

• To develop and validate a method for detecting sequential conformational changes and molten globule states during protein unfolding.
• To identify denaturation-sensitive sites in proteins.
• To analyze the unfolding of hen egg white lysozyme and horse heart cytochrome c.

Main Methods:

• Combined use of proteolysis (proteinase K) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).
• Analysis of native and guanidine hydrochloride (GuHCl)-unfolded lysozyme.
• Monitoring proteolysis products and time courses.

Main Results:

• Sequential conformational changes and molten globule states were detected during protein unfolding.
• Differences in proteolysis products and time courses were observed for native and unfolded lysozyme.
• Cytochrome c, lacking disulfide bonds, showed higher sensitivity to denaturant.
• Partially unfolded states at low GuHCl concentrations resembled molten globule states.
• Direct measurement of liberated peptide fragments identified denaturation-sensitive cleavage sites.

Conclusions:

• The combined proteolysis and MALDI-MS method is effective for studying protein unfolding dynamics.
• The method allows direct identification of sites susceptible to denaturation.
• This approach provides insights into the early stages of protein denaturation, including molten globule formation.