Investigating Lipase/Stain\nInteractions: Determining\nInterfacial Protein Conformation with Surface Spectroscopy

Conventional bulk protein structure\ndetermination methods\nare not\nsuitable for understanding the distinct and diverse interactions of\nproteins with interfaces. Notably, interfacial activation is a feature\ncommon to many lipases involving movement of a helical “lid”\nregion upon contact with a hydrophobic surface to expose the catalytic\nsite. Here we use the surface specificity of vibrational sum frequency\ngeneration spectroscopy (VSFG) spectroscopy to directly probe the\nconformation of <i>Thermomyces lanuginosus</i> lipase (TLL)\nat hydrophobic interfaces. The TLL-catalyzed reaction at the air/water\ninterface is monitored by VSFG spectroscopy, showing loss of ester\ncarbonyl modes and appearance of carboxylate stretching modes of the\nfatty acid products. Furthermore, comparison of experimental and calculated\nVSFG spectra of the amide I band of TLL allows us to discern the subtle\nstructural changes involved with lid-opening at a hydrophobic surface.\nFinally, we report a likely orientation of this lid-open state, which\ninteracts with the surface through a loop region away from the lid\nand active site. This experimental framework for probing protein structure\nand function at interfaces addresses a significant problem in protein\nscience that is not only impeding the design of better enzymes for\nbiotechnology applications but also drug discovery targeting membrane\nassociated proteins.