NMR-based Characterization of Protein Structure, Folding, and Cold Denaturation
Federizon, Jasmin Fe C.
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The research projects described in this dissertation represent commonly pursued structural biological projects: production of stable isotope labeled proteins for nuclear magnetic resonance (NMR) spectroscopy studies (Chapter 2), structural characterization of peptides using NMR (Chapter 3), employment of biophysical techniques to study protein folding (Chapter 4) as well as structural changes induced by protein cold denaturation (Chapter 5). The corresponding chapters are arranged methodically to encapsulate the process of a typical broad structural biological study. Moreover, the projects were selected to ensure the systematic training necessary to develop and hone the skills required for conducting the future dissertation research. Chapter 2 describes the production and efficient two-step purification of Bc-Csp R3E/L66E. Homogeneity of the protein preparation was assessed by SDS-PAGE, 2D-[ 15 N, 1 H]-HSQC and ESI-FT-ICR-MS. Remarkably, the level of purity turned out to be higher when compared to previous preparations with three purification steps. Chapter 3 focuses on the structural characterization of a de novo designed 16-residue cyclic peptide comprising both L- and D amino acid residues using NMR spectroscopy. The Rosetta-based design model features two short α-helices of different handedness. Though it reflects the α-helical conformational preferences detected by NMR, the de novo designed cyclic peptide is largely flexibly disordered in solution. Chapter 4 describes the investigation of the impact of 19 F labeling of Phe rings on the slow folding mutant of IFABP (G121V) by studying protein folding kinetics and thermodynamics. While the equilibrium unfolding is not perturbed by the labeling, refolding kinetics data show that fluorination has an impact on the folding rates and activation energy. Chapter 5 employed CD spectroscopy to characterize the global properties of the cold unfolding transition of Bc-Csp R3E/L66E. The CD data show that the regular secondary elements (β-strands) remain largely intact even after decreasing the temperature to –9 °C. Consistently, FRET data show that the distance for between a tyrosine and a tryptophan side chain remains approximately the same at low temperature.