Protein Structures and Kinetics at Interfaces Probed by Sum Frequency Generation Spectroscopy

Structures and kinetics of proteins at interfaces play an important role in the functions of biological organisms. During the past two decades, the development of sum frequency generation (SFG) spectroscopy has enabled the extraction of protein structural information exclusively at interfaces. However, many previous SFG studies have focused only on the amide I and N-H stretch vibrations of proteins, with information in other vibrational regions unexplored. Besides, the limitation in sample preparations has posed a challenge for characterizing native proteins at interfaces. In addition, the requirement for computational resources to simulate biomacromolecular properties has rendered it difficult to interpret SFG spectra of proteins quantitatively and accurately. This dissertation shows how SFG signals in vibrational regions other than the amide I and N-H stretch can be used to monitor the kinetics of structural changes of proteins at interfaces. Also demonstrated in this dissertation is the capability of SFG to reveal the structures and packing of amphiphilic proteins at interfaces, in conjunction with other surface characterization techniques. This dissertation also contributes to the methodology of interpreting SFG spectroscopic data with the calculation of molecular hyperpolarizabilities under various assumptions of vibrational coupling.;First, the C-H stretch vibration is used as a vibrational probe to study the self-assembly kinetics of peptide at the interface. It is demonstrated that the C-H stretch chiral SFG signals of an amphiphilic peptide (LK 7beta) can reveal its self-assembly kinetics at the air/water interface. The chiral SFG experiments combined with measurements of surface pressure point to a mechanism of the self-assembly process that involves an immediate adsorption of disordered structures followed by a lag phase before the self-assembly into chiral anti-parallel beta-sheet structures. The method of using the C-H stretch implies a general application of chiral SFG spectroscopy to study the self-assembly of bioactive, simple organic, and polymeric molecules into chiral macromolecular and supramolecular structures at interfaces. This method will shed light on problems such as protein aggregation, rational design of functional materials, and fabrication of molecular devices.;Second, SFG spectroscopy is extended to the structural study of an amphiphilic native protein with mixed secondary structures at interfaces. Expressed by a strain of gram-positive soil bacteria, this protein, known as Bs1A, is one of the major protein components in the extracellular matrix of bacterial biofilm. After being expressed and purified, the surface activity and structure of BslA at the air/water interface is characterized using SFG spectroscopy to examine its surface activity and structure. An unusually sharp amide I vibrational peak is observed, with a damping factor of ~5.25 cm-1, corresponding to ~10.5 cm-1 of full-width-at-half-maximum, which is among the sharpest amide I vibrational bands reported for native proteins. The sharp amide I peak is hypothesized to originate from ordered structure and packing of Bs1A at the interface. In conjunction with methods in surface pressure measurements, thin film X-ray reflectivity, and atomic force microscopy, it is demonstrated that BslA indeed forms extremely ordered structures at the air/water interface. The results point to the direction that BslA can form the outmost layer at the air/water or hydrophobic/water interfaces as a protective shield to contain microbial growth into colonies. The results provide insights into targeting biofilm proteins for developing antimicrobial agents and designing novel surface-active biomaterials.;Finally, ab initio quantum-chemistry calculation is performed to interpret the chiral SFG spectra of proteins and peptides with a-helical secondary structures. The study is based on a recent surprising observation made in our lab about the N-H stretch and amide I vibrations along the backbone of alpha-helical proteins. In spite of the same helical arrangement, the two molecular moieties (N-H and CO)= in amide groups have distinct visibility in chiral SFG: the N-H stretch band is prominent, while the amide I band is silent. To interpret the results, theoretical analyses at the level of quantum chemistry is performed. Specifically, molecular hyperpolarizabilities of a-helical backbone are calculated assuming different extents of vibrational coupling. The calculated hyperpolarizabilities are used to simulate the chiral SFG intensities in amide I and N-H stretch regions, which demonstrate that vibrational coupling significantly lowers the chiral SFG signal from the amide I mode. The study can provide a fundamental basis for developing chiral SFG to identify alpha-helical structures at interfaces, and establish a theoretical framework to compute molecular hyperpolarizabilities incorporating various degrees of vibrational coupling.