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A Method To Measure Protein Unfolding at an Air-Liquid Interface
Danielle L Leiske1, Ian C Shieh1, Martha Lovato Tse1
1Early Stage Pharmaceutical Development and ‡Late Stage Pharmaceutical Development, Genentech , South San Francisco, California 94080, United States.
Langmuir : the ACS Journal of Surfaces and Colloids
|September 20, 2016
Summary
Researchers developed a new method to measure protein conformation at air-liquid interfaces using Nile red fluorescence. This technique reveals time-dependent changes in protein hydrophobicity, aiding in understanding protein aggregation and folded states.
Area of Science:
- Biophysical Chemistry
- Protein Science
- Surface Science
Background:
- Proteins adsorb to hydrophobic interfaces, like the air-liquid interface.
- Surface flow can increase protein aggregation, which is often undesirable.
- Measuring protein conformation at interfaces is challenging but crucial for understanding aggregation.
Purpose of the Study:
- To develop and demonstrate a novel technique for measuring protein hydrophobicity at air-liquid interfaces.
- To assess the conformation and folded state of proteins upon adsorption.
- To investigate the time-dependent evolution of interfacial protein hydrophobicity.
Main Methods:
- Utilized the environmentally sensitive fluorophore Nile red to detect hydrophobicity.
- Applied the technique to two monoclonal antibodies (mAbs) with differing surface activities.
- Measured Nile red fluorescence at the air-liquid interface, glass-liquid interface, and in the liquid subphase.
Main Results:
- Nile red showed low background fluorescence in the liquid and at the glass-liquid interface.
- At the air-liquid interface, one mAb (mAb1) exhibited immediate fluorescence increase upon adsorption.
- Another mAb (mAb2) showed a slower evolution of fluorescence, despite constant adsorbed protein quantity, indicating time-dependent hydrophobicity changes.
Conclusions:
- Interfacial protein hydrophobicity, measurable by Nile red, correlates with protein conformation and folded state.
- The technique provides insights into the time-dependent unfolding of proteins at interfaces.
- This method offers a valuable tool for studying interfacial protein behavior with minimal sample requirements.
