Structural and Functional Studies of Peptide and Protein On Engineered Surfaces/Interfaces

Abstract

Biomolecular decorated surfaces have shown great potential in many applications ranging from antimicrobial coatings to biosensing and biofuels due to their excellent properties. The performance of such biomolecular functionalized surfaces is largely dependent on the molecular structure of surface immobilized biomolecules, the surfaces used for attachment, and the surrounding environment biomolecules are functioning in. Moreover, maintaining the functions of such biomolecular surfaces in the absence of bulk water is challenging but important for extending the applications of such surfaces to non-aqueous environment. In order to have an in-depth understanding of how such biomolecule immobilized surfaces should be designed with optimized functions, molecular level characterization needs to be done to reveal the structures of interfacial biomolecules. Here my thesis research mainly focuses on the investigations of structures (conformations and orientations) of immobilized peptides and proteins at the molecular level using sum frequency generation vibrational spectroscopy (SFG), supplemented by circular dichroism (CD) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). The relations of structures and functions of the biomolecular surfaces are elucidated. I first studied the effect of the immobilization site (e.g., N- or C-terminus) on the structure and activity of surface immobilized antimicrobial peptide (AMP) MSI-78 using a combination of SFG, CD, coarse grained MD simulation, and antibacterial testing. This peptide exhibits similar secondary structure but different orientations when immobilized with different termini, leading to varied antibacterial activity. In order to determine whether a peptide could be engineered to assume a different orientation (standing up instead of lying down), a combined coarse grained MD simulation/SFG approach was developed to design AMPs with controlled orientations after surface immobilization. To extend this research into more complicated systems, surfaces immobilized with enzymes were characterized using SFG and coarse grained MD simulation. Results show that not only can the orientation of these immobilized enzymes be controlled by selecting the surface immobilization site, but this surface orientation can dictate the enzymatic activity. The enzymatic activity is also affected by the property of the underlined surface for enzyme immobilization. With a more hydrophilic surface, a better enzymatic activity was observed. Thirdly, methods of retaining the structure and function of immobilized biomolecules in the absence of bulk water were developed. Both native secondary structure and orientation of surface immobilized biomolecules can be retained and controlled by physically attached sugar coatings and chemically co-immobilized poly-saccharide molecules. Chemically tethered sugar was found to be able to enhance the antibacterial activity of immobilized AMPs in dry conditions. Lastly, the interfacial structures of protein therapeutics adsorbed at the silicone oil surface are characterized by SFG. SFG signals contributed by both alpha helical and beta sheet structures were observed from proteins at the silicone oil surfaces. Nonionic surfactants are effective on reducing protein aggregations at such surfaces. This thesis is collaborative in nature. Prof. Neil Marsh’s group performed the enzyme engineering, and Prof. Charlie Brooks’ group carried out the simulation. The antimicrobial activity measurements were done by Prof. Chuanwu Xi’s group and Prof. Nick Abbott’s group. The CVD coatings were made by Prof. Joerg Lahann’s group. This thesis provides a detailed and systematic study of how peptides and proteins behave on abiotic surfaces in different chemical environments. Methodologies on how to retain the structure and enhance the activity of surface immobilized peptides and proteins in the absence of bulk water have been developed.