Glycosylation as a Facile Route to Control Enzyme Orientation at Interfaces

Protein glycosylation is known to impact structural and functional dynamics, yet its influence on interfacial behavior remains underexplored. Here, we systematically investigate the effects of glycosylation on the binding orientation of the <i>Thermomyces lanuginosus</i> lipase (TLL) variants at the air/water interface. Using a combination of experimental vibrational sum frequency generation (VSFG) spectroscopy and spectral calculations, we directly probe the interfacial conformation of TLL with varying degrees of glycosylation. Our findings reveal that the lid-open conformation is preferred for both glycosylated and deglycosylated forms and that the N33Q point mutation does not significantly alter binding. Additionally, high-mannose glycosylation broadens the range of preferred orientations. Complementary surface pressure measurements show similar protein concentrations across variants, suggesting that the reduced VSFG intensity for glycosylated TLL arises from an increased interfacial disorder. These results demonstrate that glycosylation can indirectly modulate protein surface interactions, suggesting a broader role for this common post-translational modification in protein interfacial binding.