Determination of Absolute Orientation of Protein α-Helices at Interfaces Using Phase-Resolved Sum Frequency Generation Spectroscopy

Understanding the structure of proteins at surfaces is key in fields such as biomaterials research, biosensor design, membrane biophysics, and drug design. A particularly important factor is the orientation of proteins when bound to a particular surface. The orientation of the active site of enzymes or protein sensors and the availability of binding pockets within membrane proteins are important design parameters for engineers developing new sensors, surfaces, and drugs. Recently developed methods to probe protein orientation, including immunoessays and mass spectrometry, either lack structural resolution or require harsh experimental conditions. We here report a new method to track the absolute orientation of interfacial proteins using phase-resolved sum frequency generation spectroscopy in combination with molecular dynamics simulations and theoretical spectral calculations. As a model system we have determined the orientation of a helical lysine-leucine peptide at the air-water interface. The data show that the absolute orientation of the helix can be reliably determined even for orientations almost parallel to the surface.