Vibrational sum frequency generation pectroscopy of proteins at interfaces
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Abstract
This thesis is about Sum Frequency Generation Spectroscopy (SFG) of proteins at different interfaces. The main goal was to obtain detailed structural information of proteins at interfaces gained by this method. To obtain protein structural information is important for the understanding of protein functions. But drawing a molecular picture of proteins at or in cell membranes remains a rarely reached holy grail in protein biophysics. Hence, finding ways for solving structures of those proteins can substantially deepen our understanding of the function of cell membranes.
The results of this thesis are divided into three sections. In the first section, the tilt angle of a membrane binding protein is determined with SFG spectroscopy. The protein IM30 was injected below a lipid monolayer, the subsequent binding was followed by SFG spectroscopy. The protein’s tilt angle was then deduced from protein to lipid peak intensity ratios.
The second section deals with the absolute orientation of peptides at interfaces. Since tilt angles of molecules measured with non-phase-resolved SFG exhibit a 180° phase uncertainty, the absolute orientation remains unknown. Phase-resolved SFG provides this missing information. Here, phase-resolved SFG was employed to study the absolute orientation of LK-peptides at the water air interface.
And the last section approaches the holy grail of structural information of membrane proteins with atomic resolution. Here, we describe an approach combining Molecular Dynamic simulations with SFG spectroscopy to predict and verify structures of membrane proteins.
Interestingly, the experimental ease and the structural information gained are working in opposite directions. The more the SFG response of a protein is canceled out due to inversion symmetry, the more information can be deduced from its SFG spectra. IM30, which consists of more than 50 parallel alpha helices, is experimentally easy to study. Yet, the spectral information is limited to a rough estimate of the overall tilt angle. On the other hand, GlpF which mainly consists of anti-parallel alpha helices exhibits an extremely low SFG intensity, but the spectral information suffices to verify MD-simulations.