Alpha-lactalbumin tertiary structure changes on hydrophobic interaction chromatography surfaces

Area of Science:

• Protein structure and dynamics
• Biophysical chemistry
• Mass spectrometry

Background:

• Alpha-lactalbumin is a model protein known for its marginal stability.
• Understanding protein behavior upon surface interaction is crucial for various applications.
• Hydrogen exchange mass spectrometry (HX-MS) is a powerful technique for probing protein structure.

Purpose of the Study:

• To investigate the structural consequences of alpha-lactalbumin adsorption onto a hydrophobic interaction chromatography (HIC) surface.
• To analyze the stability of different regions within the protein under varying conditions.
• To correlate solvent accessibility with unfolding propensity upon surface interaction.

Main Methods:

• Hydrogen exchange detected by mass spectrometry (HX-MS) was employed.
• Hydrophobic interaction chromatography (HIC) was used for protein separation.
• Proteolytic fragmentation was utilized for peptide-level HX analysis.

Main Results:

• Two distinct peaks of alpha-lactalbumin were observed after HIC, indicating different structural states.
• The less-retained peak exhibited native-like solvent exposure, while the retained peak showed partial unfolding.
• Helix C was identified as the most stable region, persisting even in the partially unfolded state.

Conclusions:

• Adsorption to the HIC surface induces structural changes in alpha-lactalbumin, leading to distinct populations.
• The stability of alpha-lactalbumin is influenced by its interaction with the HIC matrix.
• Solvent accessibility under native conditions may predict susceptibility to unfolding upon adsorption.