pH-induced changes in adsorbed β-lactoglobulin molecules measured using atomic force microscopy

Abstract

We have used atomic force microscopy (AFM) imaging and force spectroscopy to study β-lactoglobulin (β-LG) food protein molecules adsorbed onto a mica surface. In particular, we have studied the effect of in situ changes in pH on several different properties: the topographical morphology of the adsorbed β-LG molecules, adhesion of the β-LG molecules to the underlying mica substrate, and the mechanical unfolding of the β-LG molecules. In AFM images, the structure of the adsorbed protein layer was observed to change dramatically with changes in pH. This result was consistent with the mechanical unfolding of single protein molecules within the adsorbed protein layer at different pH values performed using AFM. The short rupture length (∼50 nm) measured for the fully unfolded protein at an acidic pH value of 2.5 is in good agreement with the dominant single molecule population measured previously for this pH value. Unfolding β-LG molecules from the same protein layer at a neutral value of pH = 6.8 resulted primarily in longer rupture lengths, corresponding to dimers of β-LG. AFM force–distance curves collected at pH = 9 were dominated by a large repulsion between the AFM tip and the adsorbed protein layer, which is likely due to the extended nature of the molecules because of irreversible denaturation for pH values greater than 9. This work provides a novel insight into the mechanisms of protein adsorption onto surfaces and shows that AFM force spectroscopy is a promising tool for probing in situ conformational changes in single molecules under various conditions.