Source: Dissertation Abstracts International, Volume: 75-04(E), Section: B.

Adviser: Elsa C. Y. Yan.

A fundamental understanding of biomolecules at interfaces at the molecular level is critical for a host of important phenomena in modern biology, chemistry, and medicine. However, due to the lack of effective surface-specific techniques, there is still a gap of knowledge for characterization and control of natural and artificial molecular interfaces. Developments in the surface nonlinear spectroscopy over the past two decades have provided new tools and opportunities in this broad and important field of research. This dissertation shows how surface-specific sum frequency generation spectroscopy (SFG) can be used to probe the structure, interaction, orientation, kinetics, and dynamics of proteins at various biological interfaces. This approach greatly enhances the analysis of physical and chemical properties of interfaces for biological studies.

First, a comprehensive study of model peptides and proteins using chiral SFG spectroscopy establishes a set of vibrational optical signatures of protein secondary structures at interfaces. Chiral SFG spectra are obtained for model peptides and proteins in the N-H stretching and amide I regions, and chiral vibrational signatures that are unique to alpha-helix, antiparallel beta-sheet, parallel beta-sheet, and disordered structures at interfaces. The theory that dictates the molecular mechanism of chiroptical responses from protein secondary structures in SFG spectra is summarized and a unified consideration of important factors, including molecular symmetry, vibrational coupling, and interfacial orientation. These results demonstrate that SFG is a uniquely surface-sensitive and label-free technique that is capable of unambiguously distinguishing secondary structures of proteins at interfaces. Such work has opened a new field of study that shall have long-term impact on investigation of biological interfaces.

Aided by the optical signatures of protein secondary structures, a new approach has been established for using chiral SFG spectroscopy to investigate interfacial aggregation of human islet amyloid polypeptide (hIAPP) and its orientation at membrane surfaces. Previous research proposed that the aggregation process of hIAPP causes the death of pancreatic cells and type II diabetes, but molecular-level information is lacking for the hIAPP-cell interaction and its role in causing disease. The present study uses the chiral SFG signals in amide I and N-H stretch regions to monitor the kinetics of the misfolding for the hIAPP aggregation process at membrane surfaces in situ and in real time. The resulting kinetic data elucidate the interfacial mechanism of the hIAPP aggregation process, from disordered structures to transient alpha-helical intermediates, and finally into aggregates rich in parallel beta-sheet. Collaboration with Dr. Dequan Xiao, a postdoctoral fellow in the laboratory of Professor Victor Batista, have combined chiral SFG spectroscopy and ab initio quantum chemistry calculations to show that the aggregates bind to membranes with the beta-strands oriented at 48° relative to the interface. It is likely that this orientation causes a significant disruption of the cell membrane, shedding new light on the pathogenic mechanism of type II diabetes in vivo.

In addition, the chiral SFG signals of the N-H stretching from the protein backbone have been used as a probe for monitoring proton exchange in antiparallel beta-sheets at interfaces in situ and in real-time. Upon the addition of D2O into H2O, the N-D stretch peaks gradually buildup in the chiral SFG spectra, indicating that the N-H is substituted to N-D. Similarly, upon the addition of H2O into the D2O, the N-H stretch peak also increases, but with a faster rate. Quantitative comparison of the kinetics for H-to-D and D-to-H exchange suggests the breaking of O-H/O-D bond in water is the rate-limiting step of proton exchange in protein at the air/water interface. This study offers a novel method for revealing protein structures and dynamics at interfaces.

Overall, chiral SFG has been developed into a new analytical tool for probing molecular adhesion, transport, and orientations on biological interfaces. This emerging SFG technique also will find broad benefits in other fields, such as material and environmental sciences.