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Analysis of Spin Probe Viability for Protein Structure Investigation Using Advanced EPR Techniques
Author(s) -
Ebersol Lauren,
Bokhari Aneesa,
Silakov Alexey
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.792.29
Subject(s) - electron paramagnetic resonance , site directed spin labeling , nitroxide mediated radical polymerization , chemistry , spin label , cysteine , protein structure , pulsed epr , combinatorial chemistry , biological system , biochemistry , nuclear magnetic resonance , organic chemistry , polymer , physics , spin echo , medicine , radical polymerization , radiology , biology , magnetic resonance imaging , copolymer , enzyme
An accurate depiction of protein structure and its mobility is necessary to understanding protein function and many methods exist to determine this. Obtaining protein structural characteristic 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 by 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 structures of hard‐to‐characterize proteins by combining the distance information obtained from advanced EPR techniques with mobility information obtained from room temperature continuous wave EPR (CW EPR). Many methods of spin‐labeling a protein exist; however, the viability of these specific approaches for performing structural determination techniques like DEER has not yet been explored. 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 surface‐located cysteine residues must be removed, which may not be desirable from a structural integrity standpoint. Utilizing unnatural amino acids can alleviate this issue and the spin‐label can be added to the protein via techniques such as click chemistry, a rapid cycloaddition that can occur at physiological conditions. In this work, we compare how different spin labeling techniques perform from an experimental point of view. We seek, overall, to determine a powerful and easy‐to‐use protocol for structural determination. Here, we use T4 Lysozyme as a well‐characterized protein model and compare 4‐alkyl TEMPO installed on the unnatural amino acid p‐ ‐azidophenylalanine (pAzF) with the aforementioned MTSSL/cysteine label. For this purpose, we use a cysteine‐free version of T4 Lysozyme and genetically encode either cysteine or pAzF to specific positions. The results presented 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. Finally, we compare different advanced EPR techniques (DEER, DQC, relaxation enhancement) to determine the most robust in distance predictions. Support or Funding Information The work is funded by the Pennsylvania State University startup fund of Alexey Silakov. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .