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Analysis of Spin Probe Viability for Protein Structure Investigation Using Advanced Electron Paramagnetic Resonance Techniques
Restricted (Penn State Only)
Master of Science
Date of Defense:
March 07, 2019
Alexey Silakov, Thesis Advisor
Carsten Krebs, Committee Member
site-directed spin labeling
unnatural amino acids
An accurate depiction of protein structure and its mobility is necessary to understand protein function. Owing to the importance of this problem, many methods exist to determine structures of proteins. Obtaining structural characteristics of proteins by advanced electron paramagnetic resonance (EPR) spectroscopy techniques like double electron-electron resonance (DEER) is emerging as a powerful technique that is complementary to other well-established structural characterization tools. Such studies most commonly require the ligation of “spin labels” to different sites on the protein, often via a site-specific reaction between a stable molecule containing a nitroxide radical and an amino acid residue. We seek to develop a robust set of tools for predicting the structures of hard-to-characterize proteins by combining distance information obtained from advanced EPR techniques with mobility information obtained from room temperature continuous wave EPR (CW EPR). The most common spin-labeling methodology conjugates (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL) to a protein via a disulfide bond with a cysteine residue. To achieve this conjugation site-specifically, all native solvent-exposed cysteine residues must be removed, which may not be desirable for maintaining structural integrity. Utilizing unnatural amino acids can alleviate this issue and the spin-label can be added to the protein via synthetic techniques such as click chemistry, a rapid cycloaddition that can occur under physiological conditions. In this work, we experimentally compare different spin labeling techniques. Here, we use T4 Lysozyme as a well-characterized protein model and compare traditional spin-labeling techniques via cysteine residues to those of unnatural amino acids. The results presented herein focus on the structural rigidity of the spin labels, a key factor defining the flexibility of certain areas of the protein and the certainty of the distance determination. Moreover, as different spin probes display different spectroscopic characteristics, each spin label is evaluated in terms of DEER sensitivity and efficiency.
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