NMR Spectroscopic Characterization of Proteins Large and Small: Aggregates, Oligomers, and Peptides

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful techniques for the investigation of structural, dynamic properties and interactions of biomolecules at the atomic level. In this thesis, by using biomolecular NMR spectroscopy combined with techniques such as atomic force microscopy (AFM), Thioflavin T (ThT) fluorescence, and circular dichroism (CD), we investigated biomolecules ranging from the biggest (various protein aggregates), to the smallest (an 11-amino acid residue peptide). Chapter 3, using hen egg white lysozyme and beta-lactoglobulin as model proteins, protein amyloid fibrils as well as early-stage protein aggregation was characterised through further development and the application of diffusion NMR pulse sequences. ThT fluorescence was used to show the kinetics of fibrilization, CD was used to determine the changes in protein secondary structure during protein aggregation, and CR birefringence along with TEM was used for visual conformation of the amyloid fibril. With AFM, the morphology and physical characteristics of the early stage protein aggregates and of the amyloid fibril were investigated. Finally, an in-depth application of diffusion NMR was presented using protein molecules with non-uniform sizes occurring during the protein aggregation process and also with intentionally cross-linked protein with a cross-linker BS3. Chapter 4, structural modifications of the B-Lg during thermal-denaturation at high concentration with neutral pH (pH 6.7) were investigated with 1H-13C Heteronuclear Single Quantum Correction (HSQC) NMR spectroscopy combined with size exclusion HPLC (SE-HPLC). We employed current pasteurization conditions of 85 C at high protein concentration (12% w/w) at pH 6.7, and found that the B-Lg maintained its native-like structure with minor structural changes even after such heat treatment. Lastly, in Chapter 5, the three-dimensional structure of the peptide with toll-like receptor 4 (TLR4) inhibitory property termed VIPER (Viral Inhibitory Peptide of TLR4) and its inactive mutant peptide VIPER L6AE10A were determined with various NMR spectroscopic techniques such as, 1H- 15N HSQC, 1H- 13C HSQC, Total correlated spectroscopy (TOCSY) and Nuclear Overhauser Effect Spectroscopy (NOESY). We were able to show the difference between VIPER and the mutant VIPER structure, although there were only minor side chain substitutions. Also, we compared the resulting structural ensembles of the two peptides with the 3D structure of the VIPER region in the vaccinia virus A46 protein and were able to show that as a 11 long aa peptide, VIPER still appears to retain helical-like structure, while the mutant peptide loses its helical-structure completely. This suggests that, the alpha-helical motif is important in binding with the TIR-domains. These approaches provided relatively accurate estimate of the hydrodynamic volume of large biomolecules, the native-like structure of proteins even when they are assembled as aggregates, and also structural information on the important contributing residues in a peptide for TLR4 inhabitation.