Investigating biointerfaces using sum frequency generation vibrational spectroscopy.

Abstract

Surfaces and interfaces are important and ubiquitous in the biological world. It is difficult to investigate biointerfaces with traditional surface-sensitive techniques, as few of them can obtain interface-specific chemical and structural information in situ. A nonlinear laser spectroscopic technique, sum frequency generation (SFG) vibrational spectroscopy, has been applied in this dissertation work to investigate the conformation and orientation of various biological interfacial molecules, and the interactions between them. FXII is an important blood coagulation protein that initiates the contact activation pathway. The orientation and ordering of FXII on model polymer surfaces with and without surface charges were investigated with SFG. Significant amide I signal enhancement was observed on negatively charged sulfonated polystyrene surfaces. Such enhancement is explained by the specific orientation and increased ordering of FXII molecules induced by the surface charges. This conclusion is further confirmed by results from other supplementary techniques such as attenuated total reflection-Fourier transform infrared spectroscopy and quartz crystal microbalance. Several small peptides adsorbed onto modified polymer surfaces were studied as a model system to achieve a quantitative understanding of peptide orientation at solution/polymer interfaces. It is demonstrated that SFG can distinguish between different secondary structures such as alpha-helices and beta-sheets. In addition to polymer/solution interfaces, peptides and proteins adsorbed at membrane lipid bilayer/solution interfaces were also investigated. Orientation of melittin and a peripheral membrane protein, Gbetagamma, were determined based on SFG and attenuated total reflection Fourier transform infrared spectroscopy results. The molecular structure of lipid bilayers plays a critical role in membrane biological functions. Substrate supported lipid bilayers were used as a model for cell membrane, and their interactions with various biomolecules such as melittin, antimicrobial peptides, and an antibiotic oligomer were investigated using SFG. The sensitivity of SFG allows real-time monitoring of the structural perturbation of each individual leaflet of a lipid bilayer during its interaction with biomolecules. By analyzing spectral features collected from C=O, C-D, and C-H stretching ranges, the transmembrane motion and orientation of lipid molecules, and the adsorption behavior of interacting biomolecules were acquired simultaneously, allowing the determination of the mode of action of the interacting biomolecules.