Protein-Induced Ordering of Lipid Hydrocarbon Chains at the Air–Water Interface Quantified by Sum Frequency Susceptibility Tensor Ratio Analysis

This article presents a quantitative analytical approach for calculating the orientation of lipid chains at the air-water interface upon interaction with human serum albumin (HSA) protein by using vibrational sum frequency generation (VSFG) spectroscopy combined with susceptibility tensor ratio analysis. The lipid monolayers, which are composed of dipalmitoylphosphatidylcholine (dDPPC) and dipalmitoylphosphatidylglycerol (dDPPG), serve as model lipid membranes to probe protein-lipid interactions under varying surface pressure conditions. Despite extensive experimental efforts, quantitative molecular orientation analysis of lipid chains in the presence of the protein has remained elusive. Here, we employ a susceptibility tensor ratio-based approach to extract the orientational parameters, <cosθ> and <cos³θ>. This ratio-based methodology allows us to relate the measured effective susceptibilities to the underlying molecular tilt distribution. Together, these factors allow for a quantitative estimate of the orientation of the lipid hydrocarbon chains. Our results reveal protein-induced ordering in the lipid monolayers upon interaction with HSA, which is evidenced by a reduction in the tilt angle of lipid chains with respect to surface normal. This study provides mechanistic insight into protein-lipid interactions and demonstrates the utility of theoretical VSFG analysis in quantifying interfacial molecular orientation.